The present invention relates to bioluminescence generating agents, conjugates containing such agents linked to targeting agents, and methods of use of the conjugates for visualization neoplastic or specialty tissue during invasive and non-invasive surgical procedures.
Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon (hv). Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Luminescence may be represented as follows:
A+Bxe2x86x92X*+Y
X*xe2x86x92X+hv,
where X* is an electronically excited molecule and hv represents light emission upon return of X* to a lower energy state. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction. The color of the emitted light in a bioluminescent (or chemiluminescent or other luminescent) reaction is characteristic of the excited molecule, and is independent from its source of excitation and temperature.
An essential condition for bioluminescence is the use of molecular oxygen, either bound or free in the presence of a luciferase. Luciferases, are oxygenases, that act on a substrate, luciferin, in the presence of molecular oxygen and transform the substrate to an excited state. Upon return to a lower energy level, energy is released in the form of light [for reviews see, e.g., McElroy et al. (1966) in Molecular Architecture in Cell Physiology, Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al., Chapter 7 in Chemi-and Bioluminescence, Burr, ed., Marcel Dekker, Inc. NY, pp.321-358; Hastings, J. W. in (1995) Cell Physiology:Source Book, N. Sperelakis (ed.), Academic Press, pp 665-681; Luminescence, Narcosis and Life in the Deep Sea, Johnson, Vantage Press, NY, see, esp. pp. 50-56].
Though rare overall, bioluminescence is more common in marine organisms than in terrestrial organisms. Bioluminescence has developed from as many as thirty evolutionarily distinct origins and, thus, is manifested in a variety of ways so that the biochemical and physiological mechanisms responsible for bioluminescence in different organisms are distinct. Bioluminescent species span many genera and include microscopic organisms, such as bacteria [primarily marine bacteria including Vibrio species], fungi, algae and dinoflagellates, to marine organisms, including arthropods, mollusks, echinoderms, and chordates, and terrestrial organism including annelid worms and insects.
Bioluminescence, as well as other types of chemiluminescence, is used for quantitative determinations of specific substances in biology and medicine. For example, luciferase genes have been cloned and exploited as reporter genes in numerous assays, for many purposes. Since the different luciferase systems have different specific requirements, they may be used to detect and quantify a variety of substances. The majority of commercial bioluminescence applications are based on firefly [Photinus pyralis] luciferase. One of the first and still widely used assays involves the use of firefly luciferase to detect the presence of ATP. It is also used to detect and quantify other substrates or co-factors in the reaction. Any reaction that produces or utilizes NAD(H), NADP(H) or long chain aldehyde, either directly or indirectly, can be coupled to the light-emitting reaction of bacterial luciferase.
Another luciferase system that has been used commercially for analytical purposes is the Aequorin system. The purified jellyfish photoprotein, aequorin, is used to detect and quantify intracellular Ca2+ and its changes under various experimental conditions. The Aequorin photoprotein is relatively small [xcx9c20 kDa], nontoxic, and can be injected into cells in quantities adequate to detect calcium over a large concentration range [3xc3x9710xe2x88x927 to 10xe2x88x924 M].
Because of their analytical utility, many luciferases and substrates have been studied and well-characterized and are commercially available [e.g., firefly luciferase is available from Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantly produced firefly luciferase and other reagents based on this gene or for use with this protein are available from Promega Corporation, Madison, Wis.; the aequorin photoprotein luciferase from jellyfish and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, Ga.; coelenterazine, the naturally-occurring substrate for these luciferases, is available from Molecular Probes, Eugene, Oreg.]. These luciferases and related reagents are used as reagents for diagnostics, quality control, environmental testing and other such analyses.
One difficulty encountered by surgeons during surgical procedures either for diagnosis or treatment is to find the tissue of interest. For example, during surgeries in which tumors are excised it is difficult to localize the neoplastic tissue and to be sure to remove all of it, yet not remove healthy tissue. It is also difficult to readily detect metastases, and also, for example to locate the embryo in ectopic pregnancies.
For these reasons and others, it is an object herein to provide means for visualizing neoplastic tissue and specialty tissue during surgical procedures. It is also an object herein to provide methods of detecting neoplastic and specialty tissue.
Diagnostic systems that rely on bioluminescence for visualizing tissues in situ are provided. The systems are particularly useful for visualizing and detecting neoplastic tissue and specialty tissue, such as during non-invasive and invasive procedures. Kits that provide the components of the systems and methods using the systems for visualizing the tissue are also provided. Therapeutic methods in which photosensitizing compounds are administered are also provided.
The systems include compositions containing conjugates that include a tissue specific, particularly a tumor-specific, targeting agent linked to a targeted agent, a luciferase or luciferin. The systems also include a second composition that contains the remaining components of a bioluminescence generating reaction. In some embodiments, all components, except for activators, which are provided in situ or are present in the body or tissue, are included in a single composition.
In particular, the diagnostic systems include two compositions. A first composition that contains conjugates that, in preferred embodiments, include antibodies directed against tumor antigens conjugated to a component of the bioluminescence generating reaction, a luciferase or luciferin, preferably a luciferase are provided. In certain embodiments, conjugates containing tumor-specific targeting agents are linked to luciferases or luciferins. In other embodiments, tumor-specific targeting agents are linked to microcarriers that are coupled with, preferably more than one of the bioluminescence generating components, preferably more than one luciferase molecule.
The second composition contains the remaining components of a bioluminescence generating system, typically the luciferin or luciferase substrate. In some embodiments, these components, particularly the luciferin, are linked to a protein, such as a serum albumin, or other protein carrier. The carrier and time release formulations, permit systemically administered components to travel to the targeted tissue without interaction with blood cell components, such as hemoglobin that deactivate the luciferin or luciferase. Preferred bioluminescence generating systems include the Vargula luciferase/luciferin.
In certain, the bioluminescence generating compositions are packaged in a time release formulation, such as cyclodextran, a liposome or other such vehicle, for delivery through the bloodstream to the tumor.
The compositions are useful in methods for the detection and localization of neoplastic tissue or other tissue, such as endometriotic or an ectopic pregnancy, in a mammal, particularly a human.
The conjugates permit the specific targeting of a bioluminescent agent to neoplastic tissue or other targeted tissue. Upon binding to or interaction with the tissue, when treated with an appropriate substrate (when luciferase is linked) or luciferase (when a luciferin is linked) in the presence of activators, if necessary, light is produced, thereby permitting detection and localization of neoplastic tissue or other targeted tissue. This can, for example, permit or aid in identification of neoplastic tissue, which may then be removed using noninvasive or invasive surgical procedures. In some embodiments, the conjugate may also include a chemotherapeutic agent that is delivered to the targeted tissue.
The conjugates contain one or more targeting agents [hereinafter TA] linked, either directly or via a linker, to one or more luciferases or luciferins are provided. For purposes herein, the targeting agent is any molecule that specifically interacts with a cell surface moiety that is present on a tumor cell or tissue or neoplastic cell or tissue or other targeted cell in a higher amount or concentration than on a non-tumor or non-neoplastic cell or non-targeted cell. The higher amount or concentration is any amount or concentration that permits tumor or neoplastic cells to be distinguished from non tumor or neoplastic cells or tissues using the methods and systems and compositions provided herein.
The conjugates provided herein contain the following components: (TA)n, (L)q, and (targeted agent)m in which: at least one TA moiety is linked with or without a linker (L) to at least one targeted agent, n is 1 or more, preferably 1-3, more preferably 1 or 2, but is any number whereby the resulting conjugate binds to the tumor or neoplastic cell or tissue, q is generally 1 to 4; m is 1 or more, generally 1 or 2; L refers to a linker, and the targeted agent is any agent that when treated with an activator, will produce light.
It is understood that the above description does not represent the order in which each component is linked or the manner in which each component is linked. The TA and targeted agent (or linker and targeted agent) may be linked in any order and through any appropriate linkage, as long as the resulting conjugate binds to a neoplastic cell or tissue receptor and can be visualized by the methods herein. The TA may be linked directly to the targeted agent, such as by a covalent bond, or may be linked through a linker. The TA may also be indirectly linked through a microcarrier that is coupled to the targeted agent.
The targeted agents for use herein are luciferases or photoproteins and substrates (luciferins) therefor.
The targeting agents [TAs] include any agent that will preferentially bind to a tumor or neoplastic cell or tissue compared to cell or tissue that is not a tumor or neoplastic or that target to specialty tissues such as ligaments, tendons, endometriotic tissue, ectopic pregnancies and other such tissues. Such agents include, but are not limited to tissue specific monoclonal antibodies, methotrexate and growth factors, such as FGF, EGF, HBEFG, that can be modified whereby internalization does not occur or is reduced and other such growth factors that bind to receptors that are present in greater amounts or concentrations on neoplastic or tumorous cells or tissues than non-tumorous or non-neoplastic tissues. Monoclonal antibodies specific for tumor cell surface proteins are presently preferred. Among the monoclonal antibodies, humanized monoclonal antibodies are preferred.
Methods for diagnosis and visualization of tissues in vivo or in situ are provided. Included are methods of identifying metastatic tumors or other targeted tissue during an operative procedure, typically an exploratory procedure, by the preoperative administration of a targeting agent conjugated to one of the bioluminescence-generating components and followed by the topical or local application of the final components during surgery to illuminate areas of neoplasia. In the methods provided herein, bioluminescence is used to visualize the targeted tissue in an animal during surgical procedures, such as for surgical removal.
Methods for the preparation of the conjugates and the resulting conjugates are provided. These methods include chemical conjugation methods and methods that rely on recombinant production of the conjugates. The chemical methods rely on derivatization of the targeted agent or TA with the desired linker and then reaction with a TA or targeted agent. Alternatively, the derivatized targeted agent is coupled to a microcarrier directly or through a linker and then coupled to a TA. The chemical methods of derivatization are particularly preferred. Direct linkage is presently preferred.
Presently the methods for preparation of antibody-luciferase conjugates, such as those described in U.S. Pat. No. 5,486,455 are among the preferred methods.
If a linker is used it is selected such that it does not interfere with the activity of the targeted agent upon interaction of the conjugate with a cell surface protein. Any appropriate linker known to those of skill in this art may be used. The linker may be selected to improve activity by permitting the targeted agent to react with the activating composition. In some instances the linker is selected to increase the specificity, toxicity, solubility, serum stability, and/or intracellular availability targeted moiety. In some embodiments, several linkers may be included in order to take advantage of desired properties of each linker.
Other linkers are suited for incorporation into chemically produced conjugates. Linkers that are suitable for chemically linking conjugates include disulfide bonds, thioether bonds, hindered disulfide bonds, esters, and covalent bonds between free reactive groups, such as amine and thiol groups. These bonds are produced using heterobifunctional reagents to produce reactive thiol groups on one or both of the polypeptides and then reacting the thiol groups on one polypeptide with reactive thiol groups or amine groups on the other. Other linkers include, acid cleavable linkers, such as bismaleimideothoxy propane, acid labile-transferrin conjugates and adipic acid diihydrazide, that would be cleaved in more acidic environments; photocleavable cross linkers that are cleaved by visible or UV light. Linkers particularly suitable for coupling microcarriers to TA and targeted agents are chemical linking conjugates as well as avidin/streptavidin-biotin conjugates.
Kits containing the compositions for use in the diagnostic systems are provided. In particular, the kits include a first composition that contains the conjugates, and a second that contains the activating composition, which contains any necessary activating agents, as well as the luciferase, if the targeted agent is a luciferin, or a luciferin, if the targeted agent is a luciferase.
Thus, the kits will typically include two compositions, a first composition containing the conjugate formulated for systemic administration (or in some embodiments local or topical application), and a second composition, formulated for systemic, topical or local administration depending upon the application. Instructions for administration will be included.
Methods of detecting bioluminescent neoplasia and specialty tissues for surgical removal using surgical viewing devices are also provided. Methods of detecting and surgically removing bioluminescent neoplasia and specialty tissues using these surgical viewing devices are also provided. These devices include night vision binoculars, CCD arrays, tomographs, endoscopic devices and other such devices.
Surgical instruments for detecting and surgically removing bioluminescent neoplastic and specialty tissue are provided. Methods of detecting and surgically removing bioluminescent neoplasia and specialty tissues using these surgical viewing devices are also provided. In particular, a surgical vision device, that includes: an optical detection system that is operatively associated with the image intensifier such that an image detected by the optical detection system is transmitted to the image intensifier for viewing; an image intensifier device highly sensitive to low intensity visible light; and an objective lens assembly.
Compositions and methods of using bioluminescence targeting agents, as described herein, in conjunction with photodynamic methodologies for the treatment of neoplasms are also provided. In practicing the methods, a photodynamic drug is administered prior to the administration of a targeting agent conjugated to a component of a bioluminescence generating system. Binding of the targeting agent to the targeted neoplasia and initiation of the bioluminescent reaction results in in situ production of light at the surface of the cell thereby irradiating those neoplastic tissues that uptake the photodynamic drug. Photoactivation of the drug results in death of the targeted cell.
In practicing the methods, a conjugate containing a targeting agent linked to a luciferase or luciferin is administered to an animal prior to surgery or diagnosis. During surgery or the diagnostic procedure, when the tissues can be seen, they are contacted with a composition containing the remaining components of a bioluminescence generating system. Any tissues to which the targeting agent binds will glow, thereby permitting the surgeon to identify the tissues.
In other methods, a luciferase, such as red emitting luciferase, including those from Aristostomias, A. niger, Melanocostus and Pachystomias, that produces light that can be detected through tissue is conjugated to a targeting agent and administered prior to surgery or a diagnostic procedure. A composition containing the remaining components of the bioluminescence generating system is injected either locally or systemically prior to surgery or diagnosis. When systemically administered, the components, such as the luciferin can be provided in a time release formulation, or can include other components, such as an albumin that prevent it from getting into the blood cells or that prevent interaction with other blood components. Tissues to which the tumor-specific targeting agent binds are detected using a photomultiplier or other surgical viewing instrument directly through the skin without the need for invasive surgery.
In other methods, a luciferase that produces light that can be detected through tissue, e.g., Aristostomias, is conjugated to a targeting agent and administered prior to surgery. Light is detected from the targeted tissue using a surgical instrument, such as a laparoscope or tomogram, and the image of the targeted tissue is displayed and removed by one or more invasive or non-invasive surgical techniques.
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications of referred to herein are incorporated by reference in their entirety.
As used herein, chemiluminescence refers to a chemical reaction in which energy is specifically channeled to a molecule causing it to become electronically excited and subsequently to release a photon thereby emitting visible light. Temperature does not contribute to this channeled energy. Thus, chemiluminescence involves the direct conversion of chemical energy to light energy.
As used herein, luminescence refers to the detectable EM radiation, generally, UV, IR or visible EM radiation that is produced when the excited product of an exergonic chemical process reverts to its ground state with the emission of light. Chemiluminescence is luminescence that results from a chemical reaction. Bioluminescence is chemiluminescence that results from a chemical reaction using biological molecules [or synthetic versions or analogs thereof] as substrates and/or enzymes.
As used herein, bioluminescence, which is a type of chemiluminescence, refers to the emission of light by biological molecules, particularly proteins. The essential condition for bioluminescence is molecular oxygen, either bound or free in the presence of an oxygenase, a luciferase, which acts on a substrate, a luciferin. Bioluminescence is generated by an enzyme or other protein [luciferase] that is an oxygenase that acts on a substrate luciferin [a bioluminescence substrate] in the presence of molecular oxygen and transforms the substrate to an excited state, which upon return to a lower energy level releases the energy in the form of light.
As used herein, the substrates and enzymes for producing bioluminescence are generically referred to as luciferin and luciferase, respectively. When reference is made to a particular species thereof, for clarity, each generic term is used with the name of the organism from which it derives, for example, bacterial luciferin or firefly luciferase.
As used herein, luciferase refers to oxygenases that catalyze a light emitting reaction. For instance, bacterial luciferases catalyze the oxidation of flavin mononucleotide [FMN] and aliphatic aldehydes, which reaction produces light. Another class of luciferases, found among marine arthropods, catalyzes the oxidation of Cypridina [Vargula] luciferin, and another class of luciferases catalyzes the oxidation of Coleoptera luciferin.
Thus, luciferase refers to an enzyme or photoprotein that catalyzes a bioluminescent reaction [a reaction that produces bioluminescence]. The luciferases, such as firefly and Renilla luciferases, that are enzymes which act catalytically and are unchanged during the bioluminescence generating reaction. The luciferase photoproteins, such as the aequorin photoprotein to which luciferin is non-covalently bound, are changed, such as by release of the luciferin, during bioluminescence generating reaction. The luciferase is a protein that occurs naturally in an organism or a variant or mutant thereof, such as a variant produced by mutagenesis that has one or more properties, such as thermal stability, that differ from the naturally-occurring protein. Luciferases and modified mutant or variant forms thereof are well known. For purposes herein, reference to luciferase refers to either the photoproteins or luciferases.
Thus, reference, for example, to xe2x80x9cRenilla luciferasexe2x80x9d means an enzyme isolated from member of the genus Renilla or an equivalent molecule obtained from any other source, such as from another Anthozoa, or that has been prepared synthetically.
The luciferases and luciferin and activators thereof are referred to as bioluminescence generating reagents or components. Typically, a subset of these reagents will be provided or combined with an article of manufacture. Bioluminescence will be produced upon contacting the combination with the remaining reagents. Thus, as used herein, the component luciferases, luciferins, and other factors, such as O2, Mg2+, Ca2+ are also referred to as bioluminescence generating reagents [or agents or components].
As used herein, xe2x80x9cnot strictly catalyticallyxe2x80x9d means that the photoprotein acts as a catalyst to promote the oxidation of the substrate, but it is changed in the reaction, since the bound substrate is oxidized and bound molecular oxygen is used in the reaction. Such photoproteins are regenerated by addition of the substrate and molecular oxygen under appropriate conditions known to those of skill in this art.
As used herein, bioluminescence substrate refers to the compound that is oxidized in the presence of a luciferase, and any necessary activators, and generates light. These substrates are referred to as luciferins herein, are substrates that undergo oxidation in a bioluminescence reaction. These bioluminescence substrates include any luciferin or analog thereof or any synthetic compound with which a luciferase interacts to generate light. Preferred substrates are those that are oxidized in the presence of a luciferase or protein in a light-generating reaction. Bioluminescence substrates, thus, include those compounds that those of skill in the art recognize as luciferins. Luciferins, for example, include firefly luciferin, Cypridina [also known as Vargula] luciferin [coelenterazine], bacterial luciferin, as well as synthetic analogs of these substrates or other compounds that are oxidized in the presence of a luciferase in a reaction the produces bioluminescence.
As used herein, capable of conversion into a bioluminescence substrate means susceptible to chemical reaction, such as oxidation or reduction, that yields a bioluminescence substrate. For example, the luminescence producing reaction of bioluminescent bacteria involves the reduction of a flavin mononucleotide group (FMN) to reduced flavin mononucleotide (FMNH2) by a flavin reductase enzyme. The reduced flavin mononucleotide [substrate] then reacts with oxygen [an activator] and bacterial luciferase to form an intermediate peroxy flavin that undergoes further reaction, in the presence of a long-chain aldehyde, to generate light. With respect to this reaction, the reduced flavin and the long chain aldehyde are substrates.
As used herein, bioluminescence system [or bioluminescence generating system] refers to the set of reagents required to conduct a bioluminescent reaction. Thus, the specific luciferase, luciferin and other substrates, solvents and other reagents that may be required to complete a bioluminescent reaction form a bioluminescence system. Thus a bioluminescence system refers to any set of reagents that, under appropriate reaction conditions, yield bioluminescence. Appropriate reaction conditions refers to the conditions necessary for a bioluminescence reaction to occur, such as pH, salt concentrations and temperature. In general, bioluminescence systems include a bioluminescence substrate, luciferin, a luciferase, which includes enzymes luciferases and photoproteins, and one or more activators. A specific bioluminescence system may be identified by reference to the specific organism from which the luciferase derives; for example, the Vargula [also called Cypridina] bioluminescence system (or Vargula system) includes a Vargula luciferase, such as a luciferase isolated from the ostracod, Vargula or produced using recombinant means or modifications of these luciferases. This system would also include the particular activators necessary to complete the bioluminescence reaction, such as oxygen and a substrate with which the luciferase reacts in the presence of the oxygen to produce light.
The amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations. The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
As used herein, to target a targeted agent, such as a luciferase, means to direct it to a cell that expresses a selected receptor or other cell surface protein by linking the agent to a such agent. Upon binding to or interaction with the receptor or cell surface protein the targeted agent, can be reacted with an appropriate substrate and activating agents, whereby bioluminescent light is produced and the tumorous tissue or cells distinguished from non-tumorous tissue.
As used herein, an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.
As used herein, an effective amount of a conjugate for diagnosing a disease is an amount that will result in a detectable tissue. The tissues are detected by visualization either without aid from a detector more sensitive than the human eye, or with the use of a light source to excite any fluorescent products.
As used herein, visualizable means detectable by eye, particularly during surgery under normal surgical conditions, or, if necessary, slightly dimmed light.
As used herein, pharmaceutically acceptable salts, esters or other derivatives of the conjugates include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs.
As used herein, treatment means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein.
As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).
As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
As used herein, ED50 refers to the concentration at which 50% of the cells are killed following a 72-hour incubation with a cytotoxic conjugate, such as FGF-SAP.
As used herein, ID50 refers to the concentration of a cytotoxic conjugate required to inhibit protein synthesis in treated cells by 50% compared to protein synthesis in the absence of the protein.
As used herein, targeting agent refers to an agent that specifically or preferentially targets a linked targeted agent, a luciferin or luciferase, to a neoplastic cell or tissue.
As used herein, tumor antigen refers to a cell surface protein expressed or located on the surface of tumor cells.
As used herein, neoplastic cells include any type of transformed or altered cell that exhibits characteristics typical of transformed cells, such as a lack of contact inhibition and the acquisition of tumor-specific antigens. Such cells include, but are not limited to leukemic cells and cells derived from a tumor.
As used herein, neoplastic disease is any disease in which neoplastic cells are present in the individual afflicted with the disease. Such diseases include, any disease characterized as cancer.
As used herein, metastatic tumors refers to tumors that are not localized in one site.
As used herein, specialty tissue refers to non-tumorous tissue for which information regarding location is desired. Such tissues include, for example, endometriotic tissue, ectopic pregnancies, tissues associated with certain disorders and myopathies or pathologies.
As used herein, an antibody conjugate refers to a conjugate in which the targeting agent is an antibody.
As used herein, antibody activation refers to the process whereby activated antibodies are produced. Antibodies are activated upon reaction with a linker, such as heterobifunctional reagent.
As used herein, a surgical viewing refers to any procedure in which an opening is made in the body of an animal. Such procedures include traditional surgeries and diagnostic procedures, such as laparoscopies and arthroscopic procedures.
As used herein, humanized antibodies refer to antibodies that are modified to include xe2x80x9chumanxe2x80x9d sequences of amino acids so that administration to a human will not provoke an immune response. Methods for preparation of such antibodies are known. For example, the hybridoma that expresses the monoclonal antibody is altered by recombinant DNA techniques to express an antibody in which the amino acid composition of the non-variable regions is based on human antibodies. Computer programs have been designed to identify such regions.
As used herein, ATP, AMP, NAD+ and NADH refer to adenosine triphosphate, adenosine monophosphate, nicotinamide adenine dinucleotide (oxidized form) and nicotinamide adenine dinucleotide (reduced form), respectively.
As used herein, production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
As used herein, substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
As used herein equivalent, when referring to two sequences of nucleic acids means that the two sequences in question encode the same sequence of amino acids or equivalent proteins. When xe2x80x9cequivalentxe2x80x9d is used in referring to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence with only conservative amino acid substitutions [see, e.g., Table 2, below] that do not substantially alter the activity or function of the protein or peptide. When xe2x80x9cequivalentxe2x80x9d refers to a property, the property does not need to be present to the same extent [e.g., two peptides can exhibit different rates of the same type of enzymatic activity], but the activities are preferably substantially the same. xe2x80x9cComplementary,xe2x80x9d when referring to two nucleotide sequences, means that the two sequences of nucleotides are capable of hybridizing, preferably with less than 25%, more preferably with less than 15%, even more preferably with less than 5%, most preferably with no mismatches between opposed nucleotides. Preferably the two molecules will hybridize under conditions of high stringency.
As used herein: stringency of hybridization in determining percentage mismatch is as follows:
1) high stringency: 0.1xc3x97SSPE, 0.1% SDS, 65xc2x0 C.
2) medium stringency: 0.2xc3x97SSPE, 0.1% SDS, 50xc2x0 C.
3) low stringency: 1.0xc3x97SSPE, 0.1% SDS, 50xc2x0 C.
It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.
The term xe2x80x9csubstantiallyxe2x80x9d identical or homologous or similar varies with the context as understood by those skilled in the relevant art and generally means at least 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95% identity.
As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon administration of a compound, composition or other mixture. Biological activities may be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein the biological activity of a luciferase is its oxygenase activity whereby, upon oxidation of a substrate, light is produced.
As used herein, a composition refers to a any mixture. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a combination refers to any association between two or among more items.
As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
B. Preparation of the Conjugates
The conjugates that are provided herein contain a targeting agent, such as a tissue specific or tumor specific monoclonal antibody or fragment thereof linked either directly or via a linker to a targeted agent, a luciferase (including photoproteins or luciferase enzymes) or a luciferin. The targeted agent may be coupled to a microcarrier. The linking is effected either chemically, by recombinant expression of a fusion protein in instances when the targeted agent is a protein, and by combinations of chemical and recombinant expression. The targeting agent is one that will preferentially bind to a selected tissue or cell type, such as a tumor cell surface antigen or other tissue specific antigen.
Methods for preparing conjugates are known to those of skill in the art. For example, aequorin that is designed for conjugation and conjugates containing such aequorin have been produced [see, e.g., International PCT application No. WO 94/18342; see, also Smith et al. (1995) in American Biotechnology Laboratory]. Aequorin has been conjugated to an antibody molecule by means of a sulfhydryl-reacting binding agent (Stultz et al. (1992) Use of Recombinant Biotinylated Apoaequorin from Escherichia coli. Biochemistry 31, 1433-1442). Such methods may be adapted for use herein to produce aequorin coupled to protein or other such molecules, which are useful as targeting agents. Vargula luciferase has also been linked to other molecules [see, e.g., Japanese application No. JP 5064583, Mar. 19, 1993]. Such methods may be adapted for use herein to produce aequorin coupled to protein or other such molecules, which are useful as targeting agents.
Aequorin-antibody conjugates have been employed to detect the presence of or quantitate a particular antigen in a biological sample by direct correlation to the light emitted from the bioluminescent reaction.
As an alternative, a component of the bioluminescence generating system may be modified for linkage, such as by addition of amino acid residues that are particularly suitable for linkage to the selected substrate. This can be readily effected by modifying the DNA and expressing such modified DNA to produce luciferase with additional residues at the N- or C-terminus.
Selection of the system depends upon factors such as the desired color and duration of the bioluminescence desired as well as the particular item. Selection of the targeting agent primarily depends upon the type and characteristics of neoplasia or tissue to be visualized and the setting in which visualization will be performed. For example, the luciferase isolated from Aristostomias emits red light, which is particularly beneficial for preoperative diagnosis because the red light is detectable through tissue using a photomultiplier.
1 Bioluminescence Generating Systems
A bioluminescence generating system refers to the components that are necessary and sufficient to generate bioluminescence. These include a luciferase, luciferin and any necessary co-factors or conditions. Virtually any bioluminescent system known to those of skill in the art will be amenable to use in the apparatus, systems, combinations and methods provided herein. Factors for consideration in selecting a bioluminescent-generating system, include, but are not limited to: the targeting agent used in combination with the bioluminescence; the medium in which the reaction is run; stability of the components, such as temperature or pH sensitivity; shelf life of the components; sustainability of the light emission, whether constant or intermittent; availability of components; desired light intensity; color of the light; and other such factors.
a. General Description
In general, bioluminescence refers to an energy-yielding chemical reaction in which a specific chemical substrate, a luciferin, undergoes oxidation, catalyzed by an enzyme, a luciferase. Bioluminescent reactions are easily maintained, requiring only replenishment of exhausted luciferin or other substrate or cofactor or other protein, in order to continue or revive the reaction. Bioluminescence generating reactions are well-known to those of skill in this art and any such reaction may be adapted for use in combination with articles of manufacture as described herein.
There are numerous organisms and sources of bioluminescence generating systems, and some representative genera and species that exhibit bioluminescence are set forth in the following table [reproduced in part from Hastings in (1995) Cell Physiology:Source Book, N. Sperelakis (ed.), Academic Press, pp 665-681]:
Other bioluminescent organisms contemplated for use herein are Gonadostomias, Gaussia, Watensia, Halisturia, Vampire squid, Glyphus, Mycotophids (fish), Vinciguerria, Howella, Florenciella, Chaudiodus, Melanocostus and Sea Pens.
It is understood that a bioluminescence generating system may be isolated from natural sources, such as those in the above Table, or may be produced synthetically. In addition, for uses herein, the components need only be sufficiently pure so that mixture thereof, under appropriate reaction conditions, produces a glow so that cells and tissues can be visualized during a surgical procedure.
Thus, in some embodiments, a crude extract or merely grinding up the organism may be adequate. Generally, however, substantially pure components are used. Also, components may be synthetic components that are not isolated from natural sources. DNA encoding luciferases is available [see, e.g., SEQ ID Nos. 1-13] and has been modified [see, e.g., SEQ ID Nos. 3 and 10-13] and synthetic and alternative substrates have been devised. The DNA listed herein is only representative of the DNA encoding luciferases that is available.
Any bioluminescence generating system, whether synthetic or isolated form natural sources, such as those set forth in Table 1, elsewhere herein or known to those of skill in the art, is intended for use in the combinations, systems and methods provided herein. Chemiluminescence systems per se, which do not rely on oxygenases [luciferases] are not encompassed herein.
(1) Luciferases
The targeted agents herein include luciferases or luciferins. Luciferases refer to any compound that, in the presence of any necessary activators, catalyze the oxidation of a bioluminescence substrate [luciferin] in the presence of molecular oxygen, whether free or bound, from a lower energy state to a higher energy state such that the substrate, upon return to the lower energy state, emits light. For purposes herein, luciferase is broadly used to encompass enzymes that act catalytically to generate light by oxidation of a substrate and also photoproteins, such as aequorin, that act, though not strictly catalytically [since such proteins are exhausted in the reaction], in conjunction with a substrate in the presence of oxygen to generate light. These luciferases, including photoproteins, such as aequorin, are herein also included among the luciferases. These reagents include the naturally-occurring luciferases [including photoproteins], proteins produced by recombinant DNA, and mutated or modified variants thereof that retain the ability to generate light in the presence of an appropriate substrate, co-factors and activators or any other such protein that acts as a catalyst to oxidize a substrate, whereby light is produced.
Generically, the protein that catalyzes or initiates the bioluminescent reaction is referred to as a luciferase, and the oxidizable substrate is referred to as a luciferin. The oxidized reaction product is termed oxyluciferin, and certain luciferin precursors are termed etioluciferin. Thus, for purposes herein bioluminescence encompasses light produced by reactions that are catalyzed by [in the case of luciferases that act enzymatically] or initiated by [in the case of the photoproteins, such as aequorin, that are not regenerated in the reaction] a biological protein or analog, derivative or mutant thereof.
For clarity herein, these catalytic proteins are referred to as luciferases and include enzymes such as the luciferases that catalyze the oxidation of luciferin, emitting light and releasing oxyluciferin. Also included among luciferases are photoproteins, which catalyze the oxidation of luciferin to emit light but are changed in the reaction and must be reconstituted to be used again. The luciferases may be naturally occurring or may be modified, such as by genetic engineering to improve or alter certain properties. As long as the resulting molecule retains the ability to catalyze the bioluminescent reaction, it is encompassed herein.
Any protein that has luciferase activity [a protein that catalyzes oxidation of a substrate in the presence of molecular oxygen to produce light as defined herein] may be used herein. The preferred luciferases are those that are described herein or that have minor sequence variations. Such minor sequence variations include, but are not limited to, minor allelic or species variations and insertions or deletions of residues, particularly cysteine residues. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224). Such substitutions are preferably made in accordance with those set forth in TABLE 2 as follows:
Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions. Any such modification of the polypeptide may be effected by any means known to those of skill in this art.
The luciferases may be obtained commercially, isolated from natural sources, expressed in host cells using DNA encoding the luciferase, or obtained in any manner known to those of skill in the art. For purposes herein, crude extracts obtained by grinding up selected source organisms may suffice. Since large quantities of the luciferase may be desired, isolation of the luciferase from host cells is preferred. DNA for such purposes is widely available as are modified forms thereof.
Examples of luciferases include, but are not limited to, those isolated from the ctenophores Mnemiopsis (mnemiopsin) and Beroe ovata (berovin), those isolated from the coelenterates Aequorea (aequorin), Obelia (obelin), Pelagia, the Renilla luciferase, the luciferases isolated from the mollusca Pholas (pholasin), the luciferases isolated from fish, such as Aristostomias, Pachystomias and Poricthys and from the ostracods, such as Cypridina (also referred to as Vargula). Preferred luciferases for use herein are the Aequorin protein, Renilla luciferase and Cypridina [also called Vargula] luciferase [see, e.g., SEQ ID Nos. 1, 2, and 4-13]. Also, preferred are luciferases which react to produce red and/or near infrared light. These include luciferases found in species of Aristostomias, such as A. scintillans, Pachystomias, Malacosteus, such as M. niger. 
(2) Luciferins
The substrates for the reaction or for inclusion in the conjugates include any molecule(s) with which the luciferase reacts to produce light. Such molecules include the naturally-occurring substrates, modified forms thereof, and synthetic substrates [see, e.g., U.S. Pat. Nos. 5,374,534 and 5,098,828]. Exemplary luciferins include those described herein, as well as derivatives thereof, analogs thereof, synthetic substrates, such as dioxetanes [see, e.g., U.S. Pat. Nos. 5,004,565 and 5,455,357], and other compounds that are oxidized by a luciferase in a light-producing reaction [see, e.g., U.S. Pat. Nos. 5,374,534, 5,098,828 and 4,950,588]. Such substrates also may be identified empirically by selecting compounds that are oxidized in bioluminescent reactions.
(3) Activators
The bioluminescent generating systems also require additional components discussed herein and known to those of skill in the art. All bioluminescent reactions require molecular oxygen in the form of dissolved or bound oxygen. Thus, molecular oxygen, dissolved in water or in air or bound to a photoprotein, is the activator for bioluminescence reactions. Depending upon the form of the components, other activators include, but are not limited to, ATP [for firefly luciferase], flavin reductase [bacterial systems] for regenerating FMNH2 from FMN, and Ca2+ or other suitable metal ion [aequorin].
Most of the systems provided herein will generate light when the luciferase and luciferin are mixed and exposed to air or water. The systems that use photoproteins that have bound oxygen, such as aequorin, however, will require exposure to Ca2+ [or other suitable metal ion], which can be provided in the form of an aqueous composition of a calcium salt. In these instances, addition of a Ca2+ [or other suitable metal ion] to a mixture of luciferase [aequorin] and luciferin [such as coelenterazine] will result in generation of light. The Renilla system and other Anthozoa systems also require Ca2+ [or other suitable metal ion].
If crude preparations are used, such as ground up Cypridina [shrimp] or ground fireflies, it may be necessary to add only water. In instances in which fireflies [or a firefly or beetle luciferase] are used the reaction may only require addition ATP. The precise components will be apparent, in light of the disclosure herein, to those of skill in this art or may be readily determined empirically.
It is also understood that these mixtures will also contain any additional salts or buffers or ions that are necessary for each reaction to proceed. Since these reactions are well-characterized, those of skill in the art will be able to determine precise proportions and requisite components. Selection of components will depend upon the apparatus, article of manufacture and luciferase. Various embodiments are described and exemplified herein; in view of such description, other embodiments will be apparent.
(4) Reactions
In all embodiments, all but one component, either the luciferase or luciferin, of a bioluminescence generating system will be mixed or packaged with or otherwise combined. The remaining component is conjugated to a targeting agent and is intended for administration to an animal.
Prior to a surgical procedure, the conjugate is administered via any suitable route, whereby the targeting agent binds to the targeted tissue by virtue of its specific interaction with a tissue-specific cell surface protein. During surgery the tissue is contacted, with the remaining component(s), typically by spraying the area or local injection, and any tissue to which conjugate is bound will glow. The glow should be sufficient to see under dim light or, if necessary, in the dark.
In general, since the result to be achieved is the production of light visible to the naked eye for qualitative, not quantitative, diagnostic purposes, the precise proportions and amounts of components of the bioluminescence reaction need not be stringently determined or met. They must be sufficient to produce light. Generally, an amount of luciferin and luciferase sufficient to generate a visible glow is used; this amount can be readily determined empirically and is dependent upon the selected system and selected application. Where quantitative measurements are required, more precision may be required.
For purposes herein, such amount is preferably at least the concentrations and proportions used for analytical purposes by those of skill in the such arts. Higher concentrations may be used if the glow is not sufficiently bright. Alternatively, a microcarrier coupled to more than one luciferase molecule linked to a targeting agent may be utilized to increase signal output. Also because the conditions in which the reactions are used are not laboratory conditions and the components are subject to storage, higher concentration may be used to overcome any loss of activity. Typically, the amounts are 1 mg, preferably 10 mg and more preferably 100 mg, of a luciferase per liter of reaction mixture or 1 mg, preferably 10 mg, more preferably 100 mg. Compositions may contain at least about 0.01 mg/l, and typically 0.1 mg/l, 1 mg/l, 10 mg/l or more of each component on the item. The amount of luciferin is also between about 0.01 and 100 mg/l, preferably between 0.1 and 10 mg/l, additional luciferin can be added to many of the reactions to continue the reaction. In embodiments in which the luciferase acts catalytically and does not need to be regenerated, lower amounts of luciferase can be used. In those in which it is changed during the reaction, it also can be replenished; typically higher concentrations will be selected. Ranges of concentration per liter [or the amount of coating on substrate the results from contacting with such composition] of each component on the order of 0.1 to 20 mg, preferably 0.1 to 10 mg, more preferably between about 1 and 10 mg of each component will be sufficient. When preparing coated substrates, as described herein, greater amounts of coating compositions containing higher concentrations of the luciferase or luciferin may be used.
Thus, for example, in presence of calcium, 5 mg of luciferin, such as coelenterazine, in one liter of water will glow brightly for at least about 10 to 20 minutes, depending on the temperature of the water, when about 10 mgs of luciferase, such as aequorin photoprotein luciferase or luciferase from Renilla, is added thereto. Increasing the concentration of luciferase, for example, to 100 mg/l, provides a particularly brilliant display of light.
It is understood, that concentrations and amounts to be used depend upon the selected bioluminescence generating system but these may be readily determined empirically. Proportions, particularly those used when commencing an empirical determination, are generally those used for analytical purposes, and amounts or concentrations are at least those used for analytical purposes, but the amounts can be increased, particularly if a sustained and brighter glow is desired.
b. Ctenophore and Coelenterate Systems
Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata (berovin), and coelenterates, such as Aequorea (aequorin), Obelia (obelin) and Pelagia, produce bioluminescent light using similar chemistries [see, e.g., Stephenson et al. (1981) Biochimica et Biophysica Acta 678:65-75; Hart et al. (1979) Biochemistry 18:2204-2210; International PCT Application No. WO 94/18342, which is based on U.S. application Ser. No. 08/017,116, U.S. Pat. No. 5,486,455 and other references and patents cited herein]. The Aequorin and Renilla systems are representative and are described in detail herein as exemplary and as among the presently preferred systems. The Aequorin and Renilla systems can use the same luciferin and produce light using the same chemistry, but each luciferase is different. The Aequorin luciferase aequorin, as well as, for example, the luciferases mnemiopsin and berovin, is a photoprotein that includes bound oxygen and bound luciferin, requires Ca2+ [or other suitable metal ion] to trigger the reaction, and must be regenerated for repeated use; whereas, the Renilla luciferase acts as a true enzyme because it is unchanged during the reaction and it requires dissolved molecular oxygen.
(1) The Aequorin System
The aequorin system is well known [see, e.g., Tsuji et al. (1986) xe2x80x9cSite-specific mutagenesis of the calcium-binding photoprotein aequorin,xe2x80x9d Proc. Natl. Acad. Sci. USA 83:8107-8111; Prasher et al. (1985) xe2x80x9cCloning and Expression of the cDNA Coding for Aequorin, a Bioluminescent Calcium-Binding Protein,xe2x80x9d Biochemical and Biophysical Research Communications 126:1259-1268; Prasher et al. (1986) Methods in Enzymology 133:288-297; Prasher, et al. (1987) xe2x80x9cSequence Comparisons of cDNAs Encoding for Aequorin Isotypes,xe2x80x9d Biochemistry 26:1326-1332; Charbonneau et al. (1985) xe2x80x9cAmino Acid Sequence of the Calcium-Dependent Photoprotein Aequorin,xe2x80x9d Biochemistry 24:6762-6771; Shimomura et al. (1981) xe2x80x9cResistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein,xe2x80x9d Biochem. J. 199:825-828; Inouye et al. (1989) J. Biochem. 105:473-477; Inouye et al. (1986) xe2x80x9cExpression of Apoaequorin Complementary DNA in Escherichia coli,xe2x80x9d Biochemistry 25:8425-8429; Inouye et al. (1985) xe2x80x9cCloning and sequence analysis of cDNA for the luminescent protein aequorin,xe2x80x9d Proc. Natl. Acad. Sci. USA 82:3154-3158; Prendergast, et al. (1978) xe2x80x9cChemical and Physical Properties of Aequorin and the Green Fluorescent Protein Isolated from Aequorea forskaleaxe2x80x9d J. Am. Chem. Soc. 17:3448-3453; European Patent Application 0 540 064 A1; European Patent Application 0 226 979 A2, European Patent Application 0 245 093 A1 and European Patent Application 0 245 093 B1; U.S. Pat. No. 5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat. No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQ ID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form, described in U.S. Pat. No. 5,162,227, European Patent Application 0 540 064 A1 and Sealite Sciences Technical Report No. 3 (1994), is commercially available from Sealite, Sciences, Bogart, Ga. as AQUALITE(copyright)].
This system is among the preferred systems for use herein. As will be evident, since the aequorin photoprotein includes noncovalently bound luciferin and molecular oxygen, it is suitable for storage in this form as a lyophilized powder or encapsulated into a selected delivery vehicle. The system can be encapsulated into pellets, such as liposomes or other delivery vehicles. When used, the vehicles are contacted with a composition, even tap water, that contains Ca2+ [or other suitable metal ion], to produce a mixture that glows.
(a) Aequorin and Related Photoproteins
The photoprotein, aequorin, isolated from the jellyfish, Aequorea, emits light upon the addition of Ca2+ [or other suitable metal ion]. The aequorin photoprotein, which includes bound luciferin and bound oxygen that is released by Ca2+, does not require dissolved oxygen. Luminescence is triggered by calcium, which releases oxygen and the luciferin substrate producing apoaqueorin.
The bioluminescence photoprotein aequorin is isolated from a number of species of the jellyfish Aequorea. It is a 22 kilodalton [kD] molecular weight peptide complex [see, e.g., Shimomura et al. (1962) J. Cellular and Comp. Physiol. 59:233-238; Shimomura et al. (1969) Biochemistry 8:3991-3997; Kohama et al. (1971) Biochemistry 10:4149-4152; and Shimomura et al. (1972) Biochemistry 11:1602-1608]. The native protein contains oxygen and a heterocyclic compound coelenterazine, a luciferin, [see, below] noncovalently bound thereto. The protein contains three calcium binding sites. Upon addition of trace amounts Ca2+ [or other suitable metal ion, such as strontium] to the photoprotein, it undergoes a conformational change the catalyzes the oxidation of the bound coelenterazine using the protein-bound oxygen. Energy from this oxidation is released as a flash of blue light, centered at 469 nm. Concentrations of calcium ions as low as 10xe2x88x926 M are sufficient to trigger the oxidation reaction.
Naturally-occurring apoaequorin is not a single compound but rather is a mixture of microheterogeneous molecular species. Aequoria jellyfish extracts contain as many as twelve distinct variants of the protein [see, e.g., Prasher et al. (187) Biochemistry 26:1326-1332; Blinks et al. (1975) Fed. Proc. 34:474]. DNA encoding numerous forms has been isolated [see, e.g., SEQ ID Nos. 5-9 and 13].
The photoprotein can be reconstituted [see, e.g., U.S. Pat. No. 5,023,181] by combining the apoprotein, such as a protein recombinantly produced in E. coli, with a coelenterazine, such as a synthetic coelenterazine, in the presence of oxygen and a reducing agent [see, e.g., Shimomura et al. (1975) Nature 256:236-238; Shimomura et al. (1981) Biochemistry J. 199:825-828], such as 2-mercaptoethanol, and also EDTA or EGTA [concentrations between about 5 to about 100 mM or higher for applications herein] tie up any Ca2+ to prevent triggering the oxidation reaction until desired. DNA encoding a modified form of the apoprotein that does not require 2-mercaptoethanol for reconstitution is also available [see, e.g., U.S. Pat. No. 5,093,240]. The reconstituted photoprotein is also commercially available [sold, e.g., under the trademark AQUALITE(copyright), which is described in U.S. Pat. No. 5,162,227].
The light reaction is triggered by adding Ca2+ at a concentration sufficient to overcome the effects of the chelator and achieve the 10xe2x88x926 M concentration. Because such low concentrations of Ca2+ can trigger the reaction, for use in the methods herein, higher concentrations of chelator may be included in the compositions of photoprotein. Accordingly, higher concentrations of added Ca2+ in the form of a calcium salt will be required. Precise amounts may be empirically determined. For use herein, it may be sufficient to merely add water to the photoprotein, which is provided in the form of a concentrated composition or in lyophilized or powdered form. Thus, for purposes herein, addition of small quantities of Ca2+, such as those present in phosphate buffered saline (PBS) or other suitable buffers or the moisture on the tissue to which the compositions are contacted, should trigger the bioluminescence reaction.
Numerous isoforms of the aequorin apoprotein been identified isolated. DNA encoding these proteins has been cloned, and the proteins and modified forms thereof have been produced using suitable host cells [see, e.g., U.S. Pat. Nos. 5,162,227, 5,360,728, 5,093,240; see, also, Prasher et al. (1985) Biophys. Biochem. Res. Commun. 126:1259-1268; Inouye et al. (1986) Biochemistry 25:8425-8429]. U.S. Pat. No. 5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat. No. 5,288,623; U.S. Pat. No. 5,422,266, U.S. Pat. No. 5,162,227 and SEQ ID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form is commercially available form Sealite, Sciences, Bogart, Ga. as AQUALITE(copyright)]. DNA encoding apoaequorin or variants thereof is useful for recombinant production of high quantities of the apoprotein. The photoprotein is reconstituted upon addition of the luciferin, coelenterazine, preferably a sulfated derivative thereof, or an analog thereof, and molecular oxygen [see, e.g., U.S. Pat. No. 5,023,181]. The apoprotein and other constituents of the photoprotein and bioluminescence generating reaction can be mixed under appropriate conditions to regenerate the photoprotein and concomitantly have the photoprotein produce light. Reconstitution requires the presence of a reducing agent, such as mercaptoethanol, except for modified forms, discussed below, that are designed so that a reducing agent is not required [see, e.g., U.S. Pat. No. 5,093,240].
For use herein, it is preferred aequorin is produced using DNA, such as that set forth in SEQ ID Nos. 5-13 and known to those of skill in the art or modified forms thereof. The DNA encoding aequorin is expressed in a host cell, such as E. coli, isolated and reconstituted to produce the photoprotein [see, e.g., U.S. Pat. Nos. 5,418,155, 5,292,658, 5,360,728, 5,422,266, 5,162,227].
Of interest herein, are forms of the apoprotein that have been modified so that the bioluminescent activity is greater than unmodified apoaequorin [see, e.g., U.S. Pat. No. 5,360,728, SEQ ID Nos. 10-12]. Modified forms that exhibit greater bioluminescent activity than unmodified apoaequorin include proteins having sequences set forth in SEQ ID Nos. 10-12, in which aspartate 124 is changed to serine, glutamate 135 is changed to serine, and glycine 129 is changed to alanine, respectively. Other modified forms with increased bioluminescence are also available.
For use in certain embodiments herein, the apoprotein and other components of the aequorin bioluminescence generating system are packaged or provided as a mixture, which, when desired is subjected to conditions under which the photoprotein reconstitutes from the apoprotein, luciferin and oxygen [see, e.g., U.S. Pat. No. 5,023,181; and U.S. Pat. No. 5,093,240]. Particularly preferred are forms of the apoprotein that do not require a reducing agent, such as 2-mercaptoethanol, for reconstitution. These forms, described, for example in U.S. Pat. No. 5,093,240 [see, also Tsuji et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:8107-8111], are modified by replacement of one or more, preferably all three cysteine residues with, for example serine. Replacement may be effected by modification of the DNA encoding the aequorin apoprotein, such as that set forth in SEQ ID No. 5, and replacing the cysteine codons with serine.
The photoproteins and luciferases from related species, such as Obelia are also contemplated for use herein. DNA encoding the Ca2+-activated photoprotein obelin from the hydroid polyp Obelia longissima is known and available [see, e.g., Illarionov et al. (1995) Gene 153:273-274; and Bondar et al. (1995) Biochim. Biophys. Acta 1231:29-32]. This photoprotein can also be activated by Mn2+ [see, e.g., Vysotski et al. (1995) Arch. Bioch. Biophys. 316:92-93, Vysotski et al. (1993) J. Bio-lumin. Chemilumin. 8:301-305].
In general for use herein, the components of the bioluminescence are packaged or provided so that there is insufficient metal ions to trigger the reaction. When used, the trace amounts of triggering metal ion, particularly Ca2+ is contacted with the other components. For a more sustained glow, aequorin can be continuously reconstituted or can be added or can be provided in high excess.
(b) Luciferin
The aequorin luciferin is coelenterazine and analogs therein, which include molecules having the structure [formula (I)]: 
in which R1 is CH2C6H5 or CH3; R2 is C6H5, and R3 is p-C6H4OH or CH3 or other such analogs that have activity. Preferred coelenterazine has the structure in which R1 is p-CH2C6H4OH, R2 is C6H5, and R3 is p-C6H4OH, which can be prepared by known methods [see, e.g., Inouye et al. (1975) Jap. Chem. Soc., Chemistry Lttrs. pp 141-144; and Halt et al. (1979) Biochemistry 18:2204-2210]. Among the preferred analogs, are those that are modified, whereby the spectral frequency of the resulting light is shifted to another frequency.
The preferred coelenterazine has the structure (formula (II)): 
and sulfated derivatives thereof.
The reaction of coelenterazine when bound to the aequorin photoprotein with bound oxygen and in the presence of Ca2+ can represented as follows: 
The photoprotein aequorin [which contains apoaequorin bound to a coelenterate luciferin molecule] and Renilla luciferase, discussed below, can use the same coelenterate luciferin. The aequorin photoprotein catalyses the oxidation of coelenterate luciferin [coelenterazine] to oxyluciferin [coelenteramide] with the concomitant production of blue light [lambdamax=469 nm].
Importantly, the sulfate derivative of the coelenterate luciferin [lauryl-luciferin] is particularly stable in water, and thus may be used in a coelenterate-like bioluminescent system. In this system, adenosine diphosphate (ADP) and a sulpha-kinase are used to convert the coelenterazine to the sulphated form. Sulfatase is then used to reconvert the lauryl-luciferin to the native coelenterazine. Thus, the more stable lauryl-luciferin is used in the item to be illuminated and the luciferase combined with the sulfatase are added to the luciferin mixture when illumination is desired.
Thus, the bioluminescent system of Aequorea is particularly suitable for use in the methods herein. The particular amounts and the manner in which the components are provided depends upon the type of neoplasia or specialty tissue to be visualized. This system can be provided in lyophilized form, that will glow upon addition of Ca2+. It can be encapsulated, linked to microcarriers, such as microbeads, or in as a compositions, such as a solution or suspension, preferably in the presence of sufficient chelating agent to prevent triggering the reaction. The concentration of the aequorin photoprotein will vary and can be determined empirically. Typically concentrations of at least 0.1 mg/l, more preferably at least 1 mg/l and higher, will be selected. In certain embodiments, 1-10 mg luciferin/100 mg of luciferase will be used in selected volumes and at the desired concentrations will be used.
(2) The Renilla System
Representative of coelenterate systems is the Renilla system. Renilla, also known as sea pansies, are members of the class of coelenterates Anthozoa, which includes other bioluminescent genera, such as Cavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus. Bioluminescent members of the Anthozoa genera contain luciferases and luciferins that are similar in structure [see, e.g., Cormier et al. (1973) J. Cell. Physiol. 81:291-298; see, also Ward et al. (1975) Proc. Natl. Acad. Sci. U.S.A. 72:2530-2534]. The luciferases and luciferins from each of these anthozoans crossreact with one another and produce a characteristic blue luminescence.
Renilla luciferase and the other coelenterate and ctenophore luciferases, such as the aequorin photoprotein, use imidazopyrazine substrates, particularly the substrates generically called coelenterazine [see, formulae (I) and (II), above]. Other genera that have luciferases that use a coelenterazine include: squid, such as Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia, cuttlefish, Sepiolina; shrimp, such as Oplophorus, Acanthophyra, Sergestes, and Gnathophausia; deep-sea fish, such as Argyropelecus, Yarella, Diaphus, Gonadostomias and Neoscopelus.
Renilla luciferase does not, however, have bound oxygen, and thus requires dissolved oxygen in order to produce light in the presence of a suitable luciferin substrate. Since Renilla luciferase acts as a true enzyme [i.e., it does not have to be reconstituted for further use] the resulting luminescence can be long-lasting in the presence of saturating levels of luciferin. Also, Renilla luciferase is relatively stable to heat.
Renilla luciferase, DNA encoding Renilla luciferase, and use of the DNA to produce recombinant luciferase, as well as DNA encoding luciferase from other coelenterates, are well known and available [see, e.g., SEQ ID No. 1, U.S. Pat. Nos. 5,418,155 and 5,292,658; see, also, Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126:1259-1268; Cormier (1981) xe2x80x9cRenilla and Aequorea bioluminescencexe2x80x9d in Bioluminescence and Chemiluminescence, pp. 225-233; Charbonneau et al. (1979) J. Biol. Chem. 254:769-780; Ward et al. (1979) J. Biol. Chem. 254:781-788; Lorenz et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 88:4438-4442; Hori et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Hori et al. (1975) Biochemistry 14:2371-2376; Hori et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Inouye et al. (1975) Jap. Soc. Chem. Lett. 141-144; and Matthews et al. (1979) Biochemistry 16:85-91]. The DNA encoding Renilla luciferase and host cells containing such DNA provide a convenient means for producing large quantities of the enzyme [see, e.g., U.S. Pat. Nos. 5,418,155 and 5,292,658, which describe recombinant production of Renilla luciferase and the use of the DNA to isolate DNA encoding other luciferases, particularly those from related organisms].
When used herein, the Renilla luciferase can be packaged in lyophilized form, encapsulated in a vehicle, either by itself or in combination with the luciferin substrate. Prior to use the mixture is contacted with an aqueous composition, preferably a phosphate buffered saline pH 7-8; dissolved O2 will activate the reaction. Final concentrations of luciferase in the glowing mixture will be on the order of 0.01 to 1 mg/l or more. Concentrations of luciferin will be at least about 10xe2x88x928 M, but 1 to 100 or more orders of magnitude higher to produce a long lasting bioluminescence.
In certain embodiments herein, about 1 to 10 mg, or preferably 2-5 mg, more preferably about 3 mg of coelenterazine will be used with about 100 mg of Renilla luciferase. The precise amounts, of course can be determined empirically, and, also will depend to some extent on the ultimate concentration and application. In particular, about addition of about 0.25 ml of a crude extract from the bacteria that express Renilla to 100 ml of a suitable assay buffer and about 0.005 xcexcg was sufficient to produce a visible and lasting glow [see, U.S. Pat. Nos. 5,418,155 and 5,292,658, which describe recombinant production of Renilla luciferase].
Lyophilized mixtures, and compositions containing the Renilla luciferase are also provided. The luciferase or mixtures of the luciferase and luciferin may also be encapsulated into a suitable delivery vehicle, such as a liposome, glass particle, capillary tube, drug delivery vehicle, gelatin, time release coating or other such vehicle. The luciferase may also be linked to a substrate, such as biocompatible materials.
c. Crustacean, Particularly Cyrpidina Systems
The ostracods, such as Vargula serratta, hilgendorfii and noctiluca are small marine crustaceans, sometimes called sea fireflies. These sea fireflies are found in the waters off the coast of Japan and emit light by squirting luciferin and luciferase into the water, where the reaction, which produces a bright blue luminous cloud, occurs. The reaction involves only luciferin, luciferase and molecular oxygen, and, thus, is very suitable for application herein.
The systems, such as the Vargula bioluminescent systems, are particularly preferred herein because the components are stable at room temperature if dried and powdered and will continue to react even if contaminated. Further, the bioluminescent reaction requires only the luciferin/luciferase components in concentrations as low as 1:40 parts per billion to 1:100 parts per billion, water and molecular oxygen to proceed. An exhausted system can renewed by addition of luciferin.
(1) Vargula Luciferase
The Vargula luciferase is water soluble and is among those preferred for use in the methods herein. Vargula luciferase is a 555-amino acid polypeptide that has been produced by isolation from Vargula and also using recombinant technology by expressing the DNA in suitable bacterial and mammalian host cells [see, e.g., Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571; Inouye et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:9584-9587; Johnson et al. (1978) Methods in Enzymology LVII:331-349; Tsuji et al. (1978) Methods Enzymol. 57:364-72; Tsuji (19740 Biochemistry 13:5204-5209; Japanese Patent Application No. JP 3-30678 Osaka; and European Patent Application No. EP 0 387 355 A1].
(a) Purification from Cypridina
Methods for purification of Vargula [Cypridina] luciferase are well known. For example, crude extracts containing the active can be readily prepared by grinding up or crushing the Vargula shrimp. In other embodiments, a preparation of Cypridina hilgendorfi luciferase can be prepared by immersing stored frozen C. hilgendorfi in distilled water containing, 0.5-5.0 M salt, preferably 0.5-2.0 M sodium or potassium chloride, ammonium sulfate, at 0-30xc2x0 C., preferably 0-10xc2x0 C., for 1-48 hr, preferably 10-24 hr, for extraction followed by hydrophobic chromatography and then ion exchange or affinity chromatography [TORAY IND INC, Japanese patent application JP 4258288, published Sep. 14, 1993; see, also, Tsuji et al. (1978) Methods Enzymol. 57:364-72 for other methods].
The luciferin can be isolated from ground dried Vargula by heating the extract, which destroys the luciferase but leaves the luciferin intact [see, e.g., U.S. Pat. No. 4,853,327].
(b) Preparation by Recombinant Methods
The luciferase is preferably produced by expression of cloned DNA encoding the luciferase [European Patent Application No. 0 387 355 A1; International PCT Application No. WO 95/001542; see, also SEQ ID No. 5, which sets forth the sequence from Japanese Patent Application No. JP 3-30678 and Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571] DNA encoding the luciferase or variants thereof is introduced into E. coli using appropriate vectors and isolated using standard methods.
(2) Vargula Luciferin
The natural luciferin in a substituted imidazopyrazine nucleus, such a compound of formula (III): 
Analogs thereof and other compounds that react with the luciferase in a light producing reaction also may be used.
Other bioluminescent organisms that have luciferases that can react with the Vargula luciferin include, the genera Apogon, Parapriacanthus and Porichthys.
(3) Reaction
The luciferin upon reaction with oxygen forms a dioxetanone intermediate [which includes a cyclic peroxide similar to the firefly cyclic peroxide molecule intermediate]. In the final step of the bioluminescent reaction, the peroxide breaks down to form CO2 and an excited carbonyl. The excited molecule then emits a blue to blue-green light.
The optimum pH for the reaction is about 7. For purposes herein, any pH at which the reaction occurs may be used. The concentrations of reagents are those normally used for analytical reactions or higher [see, e.g., Thompson et al. (1990) Gene 96:257-262]. Typically concentrations of the luciferase between 0.1 and 10 mg/l, preferably 0.5 to 2.5 mg/l will be used. Similar concentrations or higher concentrations of the luciferin may be used.
d. Insect Bioluminescent Systems Including Fireflies, Click Beetles, and Other Insect System
The biochemistry of firefly bioluminescence was the first bioluminescent system to be characterized [see, e.g., Wienhausen et al. (1985) Photochemistry and Photobiology 42:609-611; McElroy et al. (1966) in Molecular Architecture in cell Physiology, Hayashi et al., eds. Prentice Hall, Inc., Englewood Cliffs, N.J., pp. 63-80] and it is commercially available [e.g., from Promega Corporation, Madison, Wis., see, e.g., Leach et al. (1986) Methods in Enzymology 133:51-70, esp. Table 1]. Luciferases from different species of fireflies are antigenically similar. These species include members of the genera Photinus, Photurins and Luciola. Further, the bioluminescent reaction produces more light at 30xc2x0 C. than at 20xc2x0 C., the luciferase is stabilized by small quantities of bovine albumin serum, and the reaction can be buffered by tricine.
(1) Luciferase
DNA clones encoding luciferases from various insects and the use to produce the encoded luciferase is well known. For example, DNA clones that encode luciferase from Photinus pyralis, Luciola cruciata [see, e.g., de Wet et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:7870-7873; de We et al. (1986) Methods in Enzymology 133:3; U.S. Pat. No. 4,968,613, see, also SEQ ID No. 3] are available. The DNA has also been expressed in Saccharomyces [see, e.g., Japanese Application No. JP 63317079, published Dec. 26, 1988, KIKKOMAN CORP] and in tobacco.
In addition to the wild-type luciferase modified insect luciferases have been prepared. For example, heat stable luciferase mutants, DNA-encoding the mutants, vectors and transformed cells for producing the luciferases are available. A protein with 60% amino acid sequence homology with luciferases from Photinus pyralis, Luciola mingrelica, L. cruciata or L. lateralis and having luciferase activity is available [see, e.g., International PCT Application No. WO 95/25798]. It is more stable above 30xc2x0 C. than naturally-occurring insect luciferases and may also be produced at 37xc2x0 C. or above, with higher yield.
Modified luciferases that generate light at different wavelengths [compared with native luciferase], and thus, may be selected for their color-producing characteristics. For example, synthetic mutant beetle luciferase(s) and DNA encoding such luciferases that produce bioluminescence at a wavelength different from wild-type luciferase are known [Promega Corp, International PCT Application No. WO 95/18853, which is based on U.S. application Ser. No. 08/177,081]. The mutant beetle luciferase has an amino acid sequence differing from that of the corresponding wild-type Luciola cruciata [see, e.g., U.S. Pat. Nos. 5,182,202, 5,219,737, 5,352,598, see, also SEQ ID No.3] by a substitution(s) at one or two positions. The mutant luciferase produces a bioluminescence with a wavelength of peak intensity that differs by at least 1 nm from that produced by wild-type luciferases.
Other mutant luciferase have also been produced. Mutant luciferases with the amino acid sequence of wild-type luciferase, but with at least one mutation in which valine is replaced by isoleucine at the amino acid number 233, valine by isoleucine at 239, serine by asparagine at 286, glycine by serine at 326, histidine by tyrosine at 433 or proline by serine at 452 are known [see, e.g., U.S. Pat. Nos. 5,219,737, and 5,330,906]. The luciferases are produced by expressing DNA-encoding each mutant luciferase in E. coli and isolating the protein. These luciferases produce light with colors that differ from wild-type. The mutant luciferases catalyze luciferin to produce red [xcex609 nm and 612 nm], orange[xcex595 and 607 nm] or green [xcex 558 nm] light. The other physical and chemical properties of mutant luciferase are substantially identical to native wild type-luciferase. The mutant luciferase has the amino acid sequence of Luciola cruciata luciferase with an alteration selected from Ser 286 replaced by Asn, Gly 326 replaced by Ser, His 433 replaced by Tyr or Pro 452 replaced by Ser. Thermostable luciferases are also available [see, e.g., U.S. Pat. No. 5,229,285; see, also International PCT Application No. WO 95/25798, which provides Photinus luciferase in which the glutamate at position 354 is replaced lysine and Luciola luciferase in which the glutamate at 356 is replaced with lysine].
These mutant luciferases as well as the wild type luciferases are among those preferred herein, particularly in instances when a variety of colors are desired or when stability at higher temperatures is desired. 
Analogs of this luciferin and synthetic firefly luciferins are also known to those of skill in art [see, e.g., U.S. Pat. No. 5,374,534 and 5,098,828]. These include compounds of formula (IV) [see, U.S. Pat. No. 5,098,828]: 
in which:
R1 is hydroxy, amino, linear or branched C1-C20 alkoxy, C2-C20 alkyenyloxy, an L-amino acid radical bond via the xcex1-amino group, an oligopeptide radical with up to ten L-amino acid units linked via the xcex1-amino group of the terminal unit;
R2 is hydrogen, H2PO3, HSO3, unsubstituted or phenyl substituted linear or branched C1-C20 alkyl or C2-C20alkenyl, aryl containing 6 to 18 carbon atoms, or R3xe2x80x94C(O)xe2x80x94; and
R3 is an unsubstituted or phenyl substituted linear or branched C1-C20 alkyl or C2-C20alkenyl, aryl containing 6 to 18 carbon atoms, a nucleotide radical with 1 to 3 phosphate groups, or a glycosidically attached mono- or disaccharide, except when formula (IV) is a D-luciferin or D-luciferin methyl ester.
Modified luciferins that have been modified to produce light of shifted frequencies are known to those of skill in the art.
(3) Reaction
The reaction catalyzed by firefly luciferases and related insect luciferases requires ATP, Mg2+ as well as molecular oxygen. Luciferin must be added exogenously. Firefly luciferase catalyzes the firefly luciferin activation and the subsequent steps leading to the excited product. The luciferin reacts with ATP to form a luciferyl adenylate intermediate. This intermediate then reacts with oxygen to form a cyclic luciferyl peroxy species, similar to that of the coelenterate intermediate cyclic peroxide, which breaks down to yield CO2 and an excited state of the carbonyl product. The excited molecule then emits a yellow light; the color, however, is a function of pH. As the pH is lowered the color of the bioluminescence changes from yellow-green to red.
Different species of fireflies emit different colors of bioluminescence so that the color of the reaction will be dependent upon the species from which the luciferase is obtained. Additionally, the reaction is optimized at pH 7.8.
Addition of ATP and luciferin to a reaction that is exhausted produces additional light emission. Thus, the system, once established, is relatively easily maintained. Therefore, it is highly suitable for use herein in embodiments in which a sustained glow is desired.
e. Bacterial Systems
Luminous bacteria typically emit a continuous light, usually blue-green. When strongly expressed, a single bacterium may emit 104 to 105 photons per second. Bacterial bioluminescence systems include, among others, those systems found in the bioluminescent species of the genera Photobacterium, Vibrio and Xenorhabdus. These systems are well known and well characterized [see, e.g., Baldwin et al. (1984) Biochemistry 23:3663-3667; Nicoli et al. (1974) J. Biol. Chem. 249:2393-2396; Welches et al. (1981) Biochemistry 20:512-517; Engebrecht et al. (1986) Methods in Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology 172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70; U.S. Pat. No. 4,581,3351.
(1) Luciferases
Bacterial luciferase, as exemplified by luciferase derived from Vibrio harveyi [EC 1.14.14.3, alkanol reduced-FMN-oxygen oxidoreductase 1-hydroxylating, luminescing], is a mixed function oxidase, formed by the association of two different protein subunits xcex1 and xcex2. The xcex1-subunit has an apparent molecular weight of approximately 42,000 kD and the xcex2-subunit has an apparent molecular weight of approximately 37,000 kD [see, e.g., Cohn et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 90:102-123]. These subunits associate to form a 2-chain complex luciferase enzyme, which catalyzes the light emitting reaction of bioluminescent bacteria, such as Vibrio harveyi [U.S. Pat. No. 4,581,335; Belas et al. (1982) Science 218:791-793], Vibrio fischeri [Engebrecht et al. (1983) Cell 32:773-781; Engebrecht et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158] and other marine bacteria.
Bacterial luciferase genes have been cloned [see, e.g., U.S. Pat. No. 5,221,623; U.S. Pat. No. 4,581,335; European Patent Application No. EP 386 691 A]. Plasmids for expression of bacterial luciferase, such as Vibrio harveyi, include pFIT001 (NRRL B-18080), pPALE001 (NRRL B-18082) and pMR19 (NRRL B-18081)] are known. For example the sequence of the entire lux regulon from Vibiro fisheri has been determined [Baldwin et al. (1984), Biochemistry 23:3663-3667; Baldwin et al. (1981) Biochem. 20:512-517; Baldwin et al. (1984) Biochem. 233663-3667; see, also, e.g., U.S. Pat. Nos. 5,196,318, 5,221,623, and 4,581,335]. This regulon includes luxI gene, which encodes a protein required for autoinducer synthesis [see, e.g., Engebrecht et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158], the luxC, luxD, and luxE genes, which encode enzymes that provide the luciferase with an aldehyde substrate, and the luxA and luxB genes, which encode the alpha and beta subunits of the luciferase.
Lux genes from other bacteria have also been cloned and are available [see, e.g., Cohn et al. (1985) J. Biol. Chem. 260:6139-6146; U.S. Pat. No. 5,196,524, which provides a fusion of the luxA and luxB genes from Vibrio harveyi]. Thus, luciferase alpha and beta subunit-encoding DNA is provided and can be used to produce the luciferase. DNA encoding the xcex1 [1065 bp] and xcex2 [984 bp] subunits, DNA encoding a luciferase gene of 2124 bp, encoding the alpha and beta subunits, a recombinant vector containing DNA encoding both subunits and a transformed E. coli and other bacterial hosts for expression and production of the encoded luciferase are available. In addition, bacterial luciferases are commercially available.
(2) Luciferins
Bacterial luciferins include: 
in which the tetradecanal with reduced flavin mononucleotide are considered luciferin since both are oxidized during the light emitting reaction.
(3) Reactions
The bacterial systems require, in addition to reduced flavin, five polypeptides to complete the bioluminescent reaction: two subunits, xcex1 and xcex2, of bacterial luciferin and three units of a fatty acid reductase system complex, which supplies the tetradecanal aldehyde. Examples of bacterial bioluminescent systems useful in the apparatus and methods provided herein include those derived from Vibrio fisheri and Vibrio harveyi. One advantage to this system is its ability to operate at cold temperatures; certain surgical procedures are performed by cooling the body to lower temperatures.
Bacterial luciferase catalyzes the flavin-mediated hydroxylation of a long-chain aldehyde to yield carboxylic acid and an excited flavin; the flavin decays to ground state with the concomitant emission of blue green light [xcexmax=490 nm; see, e.g., Legocki et al. (1986) Proc. Natl. Acad. Sci. USA 81:9080; see U.S. Pat. No. 5,196,524]: 
The reaction can be initiated by contacting reduced flavin mononucleotide [FMNH2] with a mixture of the bacterial luciferase, oxygen, and a long-chain aldehyde, usually n-decyl aldehyde.
DNA encoding luciferase from the fluorescent bacterium Alteromonas hanedai is known [CHISSO CORP; see, also, Japanese application JP 7222590, published Aug. 22, 1995]. The reduced flavin mononucleotide [FMNH2; luciferin] reacts with oxygen in the presence of bacterial luciferase to produce an intermediate peroxy flavin. This intermediate reacts with a long-chain aldehyde [tetradecanal] to form the acid and the luciferase-bound hydroxy flavin in its excited state. The excited luciferase-bound hydroxy flavin then emits light and dissociates from the luciferase as the oxidized flavin mononucleotide [FMN] and water. In vivo FMN is reduced again and recycled, and the aldehyde is regenerated from the acid.
Flavin reductases have been cloned [see, e.g., U.S. Pat. No. 5,484,723; see, SEQ ID No. 14 for a representative sequence from this patent]. These as well as NAD(P)H can be included in the reaction to regenerate FMNH2 for reaction with the bacterial luciferase and long chain aldehyde. The flavin reductase catalyzes the reaction of FMN, which is the luciferase reaction, into FMNH2; thus, if luciferase and the reductase are included in the reaction system, it is possible to maintain the bioluminescent reaction. Namely, since the bacterial luciferase turns over many times, bioluminescence continues as long as a long chain aldehyde is present in the reaction system.
The color of light produced by bioluminescent bacteria also results from the participation of a protein blue-florescent protein [BFP] in the bioluminescence reaction. This protein, which is well known [see, e.g., Lee et al. (1978) Methods in Enzymology LVII: 226-234], may also be added to bacterial bioluminescence reactions in order to cause a shift in the color.
f. Other Systems
(1) Dinoflagellate Bioluminescence Generating Systems
In dinoflagellates, bioluminescence occurs in organelles termed scintillons. These organelles are outpocketings of the cytoplasm into the cell vacuole. The scintillons contain only dinoflagellate luciferase and luciferin [with its binding protein], other cytoplasmic components being somehow excluded. The dinoflagellate luciferin is a tetrapyrrole related to chlorophyll: 
or an analog thereof.
The luciferase is a 135 kD single chain protein that is active at pH 6.5, but inactive at pH 8 [see, e.g., Hastings (1981) Bioluminescence and Chemiluminescence, DeLuca et al., eds. Academic Press, NY, pp.343-360]. Luminescent activity can be obtained in extracts made at pH 8 by simply shifting the pH from 8 to 6. This occurs in soluble and particulate fractions. Within the intact scintillon, the luminescent flash occurs for xcx9c100 msec, which is the duration of the flash in vivo. In solution, the kinetics are dependent on dilution, as in any enzymatic reaction. At pH 8, the luciferin is bound to a protein [luciferin binding protein] that prevents reaction of the luciferin with the luciferase. At pH 6, however, the luciferin is released and free to react with the enzyme.
(2) Systems from Molluscs, such as Latia and Pholas
Molluscs Latia neritoides and species of Pholas are bioluminescent animals. The luciferin has the structure: 
and has been synthesized [see, e.g., Shimomura et al. (1968) Biochemistry 7:1734-1738; Shimomura et al. (1972) Proc. Natl. Acad. Sci. U.S.A. 69:2086-2089]. In addition to a luciferase and luciferin the reaction has a third component, a xe2x80x9cpurple proteinxe2x80x9d. The reaction, which can be initiated by an exogenous reducing agent is represented by the following scheme: 
XH2 is a reducing agent.
Thus for practice herein, the reaction will require the purple protein as well as a reducing agent.
(3) Earthworms and Other Annelids
Earthworm species, such as Diplocardia longa, Chaetopterus and Harmothoe, exhibit bioluminescence. The luciferin has the structure: 
The reaction requires hydrogen peroxide in addition to luciferin and luciferase. The luciferase is a photoprotein.
(4) Glow Worms
The luciferase/luciferin system from the glow worms that are found in Great Britain, and in Australian and New Zealand caves are also intended for use herein.
(5) Marine Polycheate Worm Systems
Marine polycheate worm bioluminescence generating systems, such as Phyxotrix and Chaetopterus, are also contemplated for use herein.
(6) South American Railway Beetle
The bioluminescence generating system from the South American railway beetle is also intended for use herein.
(7) Fish
Of interest herein, are luciferases and bioluminescence generating systems that generate red light. These include luciferases found in species of Aristostomias, such as A. scintillans [see, e.g., O""Day et al. (1974) Vision Res. 14:545-550], Pachystomias, Malacosteus, such as M. niger. 
Blue/green emitters include cyclthone, myctophids, hatchet fish (agyropelecus), vinciguerria, howella, florenciella, and Chauliodus.
g. Other Fluorescent Proteins
(1) Green and Blue Fluorescent Proteins
As described herein, blue light is produced using the Renilla luciferase or the Aequorea photoprotein in the presence of Ca2+ and the coelenterazine luciferin or analog thereof. This light can be converted into a green light if a green fluorescent protein (GFP) is added to the reaction. Green fluorescent proteins, which have been purified [see, e.g., Prasher et al. (1992) Gene 111:229-233] and also cloned [see, e.g., International PCT Application No. WO 95/07463, which is based on U.S. application Ser. No. 08/119,678 and U.S. application Ser. No. 08/192,274, which are herein incorporated by reference], are used by cnidarians as energy-transfer acceptors. GFPs fluoresce in vivo upon receiving energy from a luciferase-oxyluciferin excited-state complex or a Ca2+-activated photoprotein. The chromophore is modified amino acid residues within the polypeptide. The best characterized GFPs are those of Aequorea and Renilla [see, e.g., Prasher et al. (1992) Gene 111 :229-233; Hart, et al. (1979)Biochemistry 18:2204-2210]. For example, a green fluorescent protein [GFP] from Aequorea victoria contains 238 amino acids, absorbs blue light and emits green light. Thus, inclusion of this protein in a composition containing the aequorin photoprotein charged with coelenterazine and oxygen, can, in the presence of calcium, result in the production of green light. Thus, it is contemplated that GFPs may be included in the bioluminescence generating reactions that employ the aequorin or Renilla luciferases or other suitable luciferase in order to enhance or alter color of the resulting bioluminescence.
GFPs are activated by blue light to emit green light and thus may be used in the absence of luciferase and in conjunction with an external light source to illuminate neoplasia and specialty tissues, as described herein. Similarly, blue fluorescent proteins (BFPs), such as from Vibrio fischeri, Vibrio harveyi or Photobacterium phosphoreum, may be used in conjunction with an external light source of appropriate wavelength to generate blue light. (See for example, Karatani, et al., xe2x80x9cA blue fluorescent protein from a yellow-emitting luminous bacterium,xe2x80x9d Photochem. Photobiol. 55(2):293-299 (1992); Lee, et al., xe2x80x9cPurification of a blue-fluorescent protein from the bioluminescent bacterium Photobacterium phosphoreumxe2x80x9d Methods Enzymol. (Biolumin. Chemilumin.) 57:226-234 (1978); and Gast, et al. xe2x80x9cSeparation of a blue fluorescence protein from bacterial luciferasexe2x80x9d Biochem. Biophys. Res. Commun. 80(1): 14-21 (1978), each incorporated in its entirety by reference herein.) In particular, GFPs, and/or BFPs or other such fluorescent proteins may be used in the methods described herein using a targeting agent conjugate by illuminating the conjugate with light of an appropriate wavelength to cause the fluorescent proteins to fluoresce.
Such systems are particularly of interest because no luciferase is needed to activate the photoprotein. These fluorescent proteins may also be used in addition to bioluminescence generating systems to enhance or create an array of different colors.
(2) Phycobiliproteins
Phycobiliproteins are water soluble fluorescent proteins derived from cyanobacteria and eukaryotic algae [see, e.g., Apt et al. (1995) J. Mol. Biol. 238:79-96; Glazer (1982) Ann. Rev. Microbiol. 36:173-198; and Fairchild et al. (1994) J. of Biol. Chem. 269:8686-8694]. These proteins have been used as fluorescent labels in immunoassay [see, Kronick (1986) J. of Immunolog. Meth. 92:1-13], the proteins have been isolated and DNA encoding them is also available [see, e.g., Pilot et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:6983-6987; Lui et al. (1993) Plant Physiol 103:293-294; and Houmard et al. (1988) J. Bacteriol. 170:5512-5521; the proteins are commercially available from, for example, ProZyme, Inc., San Leandro, Calif.].
In these organisms, the phycobiliproteins are arranged in subcellular structures termed phycobilisomes, and function as accessory pigments that participate in photosynthetic reactions by absorbing visible light and transferring the derived energy to chlorophyll via a direct fluorescence energy transfer mechanism.
Two classes of phycobiliproteins are known based on their color: phycoerythrins (red) and phycocyanins (blue), which have reported absorption maxima between 490 and 570 nm and between 610 and 665 nm, respectively. Phycoerythrins and phycocyanins are heterogenous complexes composed of different ratios of alpha and beta monomers to which one or more class of linear tetrapyrrole chromophores are covalently bound. Particular phycobiliproteins may also contain a third xcex3-subunit which often associated with (xcex1,xcex2)6 aggregate proteins.
All phycobiliproteins contain either phycothrombilin or phycoerythobilin chromophores, and may also contain other bilins phycourobilin, cryptoviolin or the 697 nm bilin. The xcex3-subunit is covalently bound with phycourobilin which results in the 495-500 nm absorption peak of B- and R-phycoerythrins. Thus, the spectral characteristics of phycobiliproetins may be influenced by the combination of the different chromophores, the subunit composition of the apophycobiliproteins and/or the local environment effecting the tertiary and quaternary structure of the phycobiliproteins.
As described above for GFPs and BFPs, phycobiliproteins are also activated by visible light of the appropriate wavelength and, thus, may be used in the absence of luciferase and in conjunction with an external light source to illuminate neoplasia and specialty tissues, as described herein. Furthermore, the attachment of phycobiliproteins to solid support matrices is known (e.g., see U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189 and 4,867,908). As noted above, these proteins may be used in combination with other fluorescent proteins and/or bioluminescence generating systems to produce an array of colors or to provide different colors over time.
As described above, attachment of phycobiliproteins to solid support matrices is known (e.g., see U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189 and 4,867,908). Therefore, phycobiliproteins may be coupled to microcarriers coupled to one or more components of the bioluminescent reaction, preferably a luciferase, to convert the wavelength of the light generated from the bioluminescent reaction. Microcarriers coupled to one or more phycobiliproteins may be used in any of the methods provided herein.
The conversion of blue or green light to light of a longer wavelength, i.e., red or near infra-red, is particularly preferred for the visualization of deep neoplasias or specialty tissues using a laparoscope or computer tomogram imaging system, as described herein.
Thus, when a change in the frequency of emitted light is desired, the phycobiliprotein, or other spectral shifter, such as synthetic fluorochrome, green fluorescent proteins, red fluorescent proteins, and substrates altered chemically or enzymatically to cause shifts in frequency of emission can be included with the bioluminescent generating components.
2. Linkers
Any linker known to those of skill in the art may be used herein. Other linkers are suitable for incorporation into chemically produced conjugates. Linkers that are suitable for chemically linked conjugates include disulfide bonds, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups. These bonds are produced using heterobifunctional reagents to produce reactive thiol groups on one or both of the polypeptides and then reacting the thiol groups on one polypeptide with reactive thiol groups or amine groups to which reactive maleimido groups or thiol groups can be attached on the other. Other linkers include, acid cleavable linkers, such as bismaleimideothoxy propane, acid labile-transferrin conjugates and adipic acid diihydrazide, that would be cleaved in more acidic intracellular compartments; cross linkers that are cleaved upon exposure to UV or visible light and linkers, such as the various domains, such as CH1, CH2, and CH3, from the constant region of human IgG, (see, Batra et al. (1993) Molecular Immunol. 30:379-386). In some embodiments, several linkers may be included in order to take advantage of desired properties of each linker.
Chemical linkers and peptide linkers may be inserted by covalently coupling the linker to the TA and the targeted agent. The heterobifunctional agents, described below, may be used to effect such covalent coupling. Peptide linkers may also be linked by expressing DNA encoding the linker and TA, linker and targeted agent, or linker, targeted agent and TA as a fusion protein.
Flexible linkers and linkers that increase solubility of the conjugates are contemplated for use, either alone or with other linkers are contemplated herein.
Numerous heterobifunctional cross-linking reagents that are used to form covalent bonds between amino groups and thiol groups and to introduce thiol groups into proteins, are known to those of skill in this art (see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog and Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such reagents; see, also, e.g., Cumber et al. (1992) Bioconjugate Chem. 3:397-401; Thorpe et al. (1987) Cancer Res. 47:5924-5931; Gordon et al. (1987) Proc. Natl. Acad Sci. 84:308-312; Walden et al. (1986) J. Mol. Cell Immunol. 2:191-197; Carlsson et al. (1978) Biochem. J. 173:723-737; Mahan et al. (1987) Anal. Biochem. 162:163-170; Wawryznaczak et al. (1992) Br. J. Cancer 66:361-366; Fattom et al. (1992) Infection and Immun. 60:584-589). These reagents may be used to form covalent bonds between the TA and targeted agent. These reagents include, but are not limited to: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; disulfide linker); sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP); succinimidyloxycarbonyl-xcex1-methyl benzyl thiosulfate (SMBT, hindered disulfate linker); succinimidyl 6-[3-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP); sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC); succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindered disulfide bond linker); sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3xe2x80x2-dithiopropionate (SAED); sulfo-succinimidyl 7-azido-4-methylcoumarin-3-acetate (SAMCA); sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)toluamido]-hexanoate (sulfo-LC-SMPT); 1,4-di-[3xe2x80x2-(2xe2x80x2-pyridyldithio)propionamido]butane (DPDPB); 4-succinimidyloxycarbonyl-xcex1-methyl-xcex1-(2-pyridylthio)toluene (SMPT, hindered disulfate linker);sulfosuccinimidyl6[xcex1-methyl-xcex1-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS); N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker); sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB); succinimidyl4(p-maleimidophenyl)butyrate (SMPB); sulfosuccinimidyl4-(p-maleimidophenyl)butyrate (sulfo-SMPB); azidobenzoyl hydrazide (ABH).
Acid cleavable linkers, photocleavable and heat sensitive linkers may also be used, particularly where it may be necessary to cleave the targeted agent to permit it to be more readily accessible to reaction. Acid cleavable linkers include, but are not limited to, bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see, e.g., Fattom et al. (1992) Infection and Immun. 60:584-589) and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway (see, e.g., Welhxc3x6ner et al. (1991) J. Biol. Chem. 266:4309-4314).
Photocleavable linkers are linkers that are cleaved upon exposure to light (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which linkers are herein incorporated by reference), thereby releasing the targeted agent upon exposure to light. Photocleavable linkers that are cleaved upon exposure to light are known (see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, which describes the use of a nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al. (1989) Makromol. Chem 190:69-82, which describes water soluble photocleavable copolymers, including hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer and methylrhodamine copolymer; Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which describes a cross-linker and reagent that undergoes photolytic degradation upon exposure to near UV light (350 nm); and Senter et al. (1985) Photochem. Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl chloride cross linking reagents that produce photocleavable linkages), thereby releasing the targeted agent upon exposure to light. Such linkers would have particular use in treating dermatological or ophthalmic conditions that can be exposed to light using fiber optics. After administration of the conjugate, the eye or skin or other body part can be exposed to light, resulting in release of the targeted moiety from the conjugate. Such photocleavable linkers are useful in connection with diagnostic protocols in which it is desirable to remove the targeting agent to permit rapid clearance from the body of the animal.
3. Targeting Agents
Targeting agents include any agent that will interact with and localize the targeted agent cells in a tumor or specialized tissue [targeted tissue]. Such agents include any agent that specifically interacts with a cell surface protein or receptor that is present at sufficiently higher concentrations or amounts on the targeted tissue, whereby, when contacted with an appropriate bioluminescence generating reagent and activators. These agents include, but are not limited to, growth factors, preferentially modified to not internalize, methotrexate, and antibodies, particularly, antibodies raised against tumor specific antigens. A plethora of tumor-specific antigens have been identified from a number of human neoplasias. Among the antigens suitable for use in raising antibodies are those set forth in Table 3. below.
Anti-tumor Antigen Antibodies
Polyclonal and monoclonal antibodies may be produced against selected antigens. Alternatively, many such antibodies are presently available. An exemplary list of antibodies and the tumor antigen for which each has been directed against is provided in Table 3. It is contemplated that any of the antibodies listed may be conjugated with a bioluminescence generating component following the methods provided herein.
Among the preferred antibodies for use in the methods herein are those of human origin or, more preferably, are humanized monoclonal antibodies. These are preferred for diagnosis of humans.
Any method for linking proteins may be used. For example, methods for linking a luciferase to an antibody is described in U.S. Pat. No. 5,486,455. As noted above, the targeting agent and luciferin or luciferase may be linked directly, such as through covalent bonds, i.e., sulfhyryl bonds or other suitable bonds, or they may be linked through a linker. There may be more than one luciferase or luciferin per targeting agent, or more than one targeting agent per luciferase or luciferin.
Alternatively, an antibody, or F(Ab)2 antigen-binding fragment thereof or other protein targeting agent may be fused (directly or via a linking peptide) to the luciferase using recombinant DNA technology. For example, the DNA encoding any of the anti-tumor antibodies of Table 3 may be ligated in the same translational reading frame to DNA encoding any of the above-described luciferases, e.g., SEQ ID NOs. 1-14 and inserted into an expression vector. The DNA encoding the recombinant antibody-luciferase fusion may be introduced into an appropriate host, such as bacteria or yeast, for expression.
C. Formulation and Administration of the Compositions for Use in the Diagnostic Systems
In most embodiments, the components of the diagnostic systems provided herein are formulated into two compositions: a first composition containing the conjugate; and a second composition containing the remaining components of the bioluminescence generating system. The compositions are formulated in any manner suitable for administration to an animal, particularly a mammal, and more particularly a human. Such formulations include those suitable for topical, local, enteric, parenteral, intracystal, intracutaneous, intravitreal, subcutaneous, intramuscular, or intraveneous administration.
For example, the conjugates, which in preferred embodiments, are a targeting agent linked to a luciferase (or photoprotein) are formulated for systemic or local administration. The remaining components are formulated in a separate second composition for topical or local application. The second composition will typically contain any other agents, such as spectral shifters that will be included in the reaction. It is preferred that the components of the second composition are formulated in a time release manner or in some other manner that prevents degradation and/or interaction with blood components.
As noted above, the conjugates either contain a luciferase or luciferin and a targeting agents. The preferred conjugates are formed between a targeting agent and a luciferase or photoprotein. The conjugates may be formulated into pharmaceutical compositions suitable for topical, local, intravenous and systemic application. Effective concentrations of one or more of the conjugates are mixed with a suitable pharmaceutical carrier or vehicle. The concentrations or amounts of the conjugates that are effective requires delivery of an amount, upon administration, that results in a sufficient amount of targeted moiety linked to the targeted cells or tissue whereby the cells or tissue can be visualized during the surgical procedure. Typically, the compositions are formulated for single dosage administration. Effective concentrations and amounts may be determined empirically by testing the conjugates in known in vitro and in vivo systems, such as those described here; dosages for humans or other animals may then be extrapolated therefrom.
Upon mixing or addition of the conjugate(s) with the vehicle, the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. The effective concentration is sufficient for targeting a sufficient amount of targeted agent to the site of interest, whereby when combined with the remaining reagents during a surgical procedure the site will glow. Such concentration or amount may be determined based upon in vitro and/or in vivo data, such as the data from the mouse xenograft model for tumors or rabbit ophthalmic model. If necessary, pharmaceutically acceptable salts or other derivatives of the conjugates may be prepared.
Pharmaceutical carriers or vehicles suitable for administration of the conjugates provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the conjugates may be formulated as the sole pharmaceutically ingredient in the composition or may be combined with other active ingredients.
The conjugates can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Intravenous or local administration is presently preferred. Tumors and vascular proliferative disorders, will typically be visualized by systemic, intradermal or intramuscular, modes of administration.
The conjugate is included in the pharmaceutically acceptable carrier in an amount sufficient to produce detectable tissue and to not result in undesirable side effects on the patient or animal. It is understood that number and degree of side effects depends upon the condition for which the conjugates are administered. For example, certain toxic and undesirable side effects are tolerated when trying to diagnose life-threatening illnesses, such as tumors, that would not be tolerated when diagnosing disorders of lesser consequence.
The concentration of conjugate in the composition will depend on absorption, inactivation and excretion rates thereof, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. Typically an effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-1000 xcexcg/ml, preferably 50-100 xcexcg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 100-2000 mg of conjugate, depending upon the conjugate selected, per kilogram of body weight per day. Typically, for intravenous administration a dosage of about between 0.05 and 1 mg/kg should be sufficient. Local application for, such as visualization of ophthalmic tissues or local injection into joints, should provide about 1 ng up to 1000 xcexcg, preferably about 1 xcexcg to about 100 xcexcg, per single dosage administration. It is understood that the amount to administer will be a function of the conjugate selected, the indication, and possibly the side effects that will be tolerated. Dosages can be empirically determined using recognized models.
The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of administration is a function of the disease condition being diagnosed and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material.
If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
The conjugates may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylacetic acid and others. These are particularly useful for application to the eye for ophthalmic indications following or during surgery in which only a single administration is possible. Methods for preparation of such formulations are known to those skilled in the art.
The conjugates may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7, with appropriate salts. The ophthalmic compositions may also include additional components, such as hyaluronic acid. The conjugates may be formulated as aerosols for topical application (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923).
Also, the compositions for activation of the conjugate in vivo during surgical procedures may be formulated as an aerosol. These compositions contain the activators and also the remaining bioluminescence generating agent, such as luciferin, where the conjugate targets a luciferase, or a luciferase, where the conjugate targets a luciferin, such as coelenterazine.
If oral administration is desired, the conjugate should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.
When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The conjugates can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as cis-platin for treatment of tumors.
Finally, the compounds may be packaged as articles of manufacture containing packaging material, one or more conjugates or compositions as provided herein within the packaging material, and a label that indicates the indication for which the conjugate is provided.
The second composition will include the remaining components of the bioluminescence generating reaction. In preferred embodiments in which these components are administered systemically, the remaining components include the luciferin or substrate, and optionally additional agents, such as spectral shifters. These components, such as the luciferin, can be formulated as described above for the conjugates. In some embodiments, the luciferin or luciferase in this composition will be linked to a protein carrier or other carrier to prevent degradation or dissolution into blood cells or other cellular components.
For embodiments, in which the second composition is applied locally or topically, they can be formulated in a spray or aerosol or other suitable means for local or topical application.
In certain embodiments described herein, all components, except an activator are formulated together, such as by encapsulation in a time release formulation that is targeted to the tissue. Upon release the composition will have been localized to the desired site, and will begin to glow.
In practice, the two compositions can be adminstered simultaneously or sequentially. Typically, the first composition, which contains the conjugate is adminstered first, generally an hour or two before the surgery, and the second composition is then adminstered, either pre-operatively or during surgery.
D. Practice of the Reactions in Combination with Targeting Agents
The particular manner in which each bioluminescence system will be combined with a selected targeting agent will be a function of the agent and the neoplasia or tissue to be visualized. In general, however, a luciferin or luciferase, of the reaction will be conjugated to the targeting agent, administered to an animal prior to surgery. During the surgery, the tissues of interest are contacted with the remaining component(s) of a bioluminescence generating system. Any tissue to which or with which the targeting agent reacts will glow.
Any color of visible light produced by a bioluminescence generating system is contemplated for use in the methods herein. Preferably the visible light is a combination of blue, green and/or red light of varying intensities and wavelengths. For visualizing neoplasia or specialty tissues through mammalian tissues or tumors deeply embedded in tissue, longer wavelengths of visible light, i.e., red and near infrared light, is preferred because wavelengths of near infrared light of about 700-1300 nm are known to penetrate soft tissue and bone [e.g., see U.S. Pat. No. 4,281,645].
In the simplest embodiments, the targeting agent conjugate may be visualized by adding one or more of the bioluminescence generating components in crude mixture. For example, the organisms can be ground up and dried and administered topically. Light will be emitted by ground up fireflies when mixed with water and ATP. Light will also be emitted by combining ground up Vargula shrimp and adding water, preferably cool water [room temperature or lower]. The only caveat is that the water must not be too hot; high temperatures destroy activity of the luciferases.
Generally, the remaining bioluminescence generating components will be formulated in a vehicle suitable for topical administration to tissues during surgery and will be applied as an aerosol. Alternatively, they can be injected into the tissue or administered intravenously.
In other embodiments, the conjugate can be applied to the tissues during surgery, such as by spraying a sterile solution over the tissues, followed by application of the remaining components. Tissues that express the targeted antigen will glow.
The reagents may be provided in compositions, such as suspensions, as powders, as pastes or any in other suitable sterile form. They may be provided as sprays, aerosols, or in any suitable form. The reagents may be linked to a matrix, particularly microbeads suitable for in vivo use and of size that they pass through capillaries. Typically all but one or more, though preferably all but one, of the components necessary for the reaction will be mixed and provided together; reaction will be triggered contacting the mixed component(s) with the remaining component(s), such as by adding Ca2+, FMN with reductase, FMNH2, ATP, air or oxygen.
In preferred embodiments the luciferase or luciferase/luciferin, such as the aequorin photoprotein, will be provided in combination with the targeting agent before administration to the patient. The targeting agent conjugate will then be contacted in vivo with the remaining components. As will become apparent herein, there are a multitude of ways in which each system may be combined with a selected targeting agent.
E. Kits and Compositions
Kits containing the diagnostic systems are provided. The kits comprise compositions containing the conjugates and remaining bioluminescence generating system components. The first composition in the kit typically contains the targeting agent conjugated to a luciferase (i.e., a luciferase or photoprotein). The second composition, contains at least the luciferin (substrate). Both compositions are formulated for systemic, local or topical application to a mammal. In alternative embodiments, the first composition contains the luciferin linked to a targeting agent, and the second composition contains the luciferase.
In general, the packaging is non-reactive with the compositions contained therein and where needed should exclude water and or air to the degree those substances are required for the luminescent reaction to proceed.
Diagnostic applications may require specific packaging. The bioluminescence generating reagents may be provided in pellets, encapsulated as micro or macro-capsules, linked to matrices, preferably biocompatible, more preferably biodegradable matrices, and included in or on articles of manufacture, or as mixtures in chambers within an article of manufacture or in some other configuration. For example, a composition containing luciferase conjugate will be provided separately from, and for use with, a separate composition containing a bioluminescence substrate and bioluminescence activator.
Similarly, the luciferase or luciferin may be provided in a composition that is a mixture, suspension, solution, powder, paste or other suitable composition separately from or in combination with the remaining components, but in the absence of an activating component. Upon contacting the conjugate, which has been targeted to a selected tissue, with this composition the reaction commences and the tissue glows. The luciferase and bioluminescence substrate, for example, are packaged to exclude water and/or air, the bioluminescence activator. Upon administration and release at the targeted site, the reaction with salts or other components at the site, including air in the case of surgical procedures, will activate the components.
1. Dispensing and Packaging Apparatus for Combination with the Bioluminescent System Components
The kits may include a bioluminescent apparatus systems provided herein are bioluminescence [or bioluminescent] systems in combination with dispensing or packaging apparatus. The bioluminescence systems, described in detail elsewhere herein, include three components: a bioluminescence substrate [e.g., a luciferin], a luciferase [e.g., a luciferase or photoprotein], and a bioluminescence activator or activators [e.g., molecular oxygen or Ca2+]. The dispensing and packaging apparatus are configured to keep at least one of the three components separate from the other two components, until generation of bioluminescence is desired. Detailed descriptions of such apparatus are described in copending, commonly owned U.S. application Ser. Nos. 08/757,046 and 08/597,274, which are incorporated by reference herein.
Two of the components will be mixed to form a suitable composition for targeting to tissues, and the third, will be administered when visualization or detection is desired.
In general, the dispensing and packaging apparatus are non-reactive with the bioluminescent system components contained therein and can exclude moisture, air or other activators, such as O2 or Ca2+, or in some manner keep all necessary components that are required for the bioluminescent reaction to come into contact until desired.
2. Capsules, Pellets, Liposomes, Endosomes, Vacuoles, Micronized Particles
In certain embodiments sequestering of the components of one of the compositions from the environment prior to use or provision of the components in particulate form, such as microparticles, may be necessary. Examples of suitable means for such use include encapsulating bioluminescent generating system components in one or micro- [up to about 100 xcexcm in size] or macroparticles [larger than 100 xcexcM] of material that permits release of the contents, such as by diffusion or by dissolution of the encapsulating material. Microparticles to which a plurality of conjugates can be linked are among the preferred embodiments. The microparticles are biocompatible and preferably of a size that can pass through capillary walls.
Liposomes and other encapsulating vehicles [see, e.g., U.S. Pat. No. 4,525,306, which describes encapsulation of compounds in gelatin; U.S. Pat. Nos. 4,021,364, 4,225,581, 4,269,821, 4,322,311, 4,324,683, 4,329,332, 4,525,306, 4,963,368 describe encapsulation of biologically active materials in various polymers] known to those of skill in the art, including those discussed herein and known to those of skill in the art [such as soluble paper, see U.S. Pat. No. 3,859,125].
a. Encapsulating Vehicles in General
All components of the bioluminescence generating system, except for the oxygen or water or Ca2+, depending upon the selected system can be incorporated into encapsulating material, such as liposomes, that protect the contents from the environment until placed into conditions that cause release of the contents into the environment. Encapsulating material contemplated for use herein includes liposomes and other such materials used for encapsulating chemicals, such as drug delivery vehicles.
b. Encapsulating Vehicles -liposomes
For example, liposomes that dissolve and slowly release the components into the medium, such as the blood, which contains dissolved oxygen or Ca 2+ or even ATP for the luciferase system are contemplated herein. They can be formulated in compositions, such as solutions, suspensions, gels, lotions, creams, and ointments, for topical application, such as procedures for diagnosing or visualizing melanomas. Liposomes and other slow release encapsulating compositions are well known and can be adapted for use in for slow release delivery of bioluminescence generating components. Typically the luciferin and luciferase will be encapsulated in the absence of oxygen or Ca2+ or ATP or other activating component. Upon release into the environment or medium containing this component at a suitable concentration, the reaction will proceed and a glow will be produced. Generally the concentrations of encapsulated components should be relatively high, perhaps 0.1-1 mg/ml or more, to ensure high enough local concentrations upon release to be visible.
Liposomes or other sustained release delivery system that are formulated in an ointment or sustained release topical vehicle, for example, would be suitable for use in a body paint, lotion. Those formulated as a suspension would be useful as a spray. Numerous ointments and suitable liposome formulations are known [see, e.g., Liposome Technology, Targeted Drug Delivery and Biological Interaction, vol. III, G. Gregoriadis ed., CRC Press, Inc., 1984; U.S. Pat. Nos. 5,470,881; 5,366,881; 5,296,231; 5,272,079; 5,225,212; 5,190,762; 5,188,837; 5,188,837; 4,921,757; 4,522,811]. For example, an appropriate ointment vehicle would contain petrolatum, mineral oil and/or anhydrous liquid lanolin. Sustained release vehicles such as liposomes, membrane or contact lens delivery systems, or gel-forming plastic polymers would also be suitable delivery vehicles. Liposomes for topical delivery are well known [see, e.g., U.S. Pat. No. 5,296,231; Mezei et al. (1980) xe2x80x9cLiposomes -A selective drug delivery system for the topical route of administration, I. lotion dosage formxe2x80x9d Life Sciences 26:1473-1477; Mezei et al. (1981) xe2x80x9cLiposomes -A selective drug delivery system for the topical route of administration: gel dosage formxe2x80x9d Journal of Pharmacy and Pharmacology 34:473-474; Gesztes et al. (1988) xe2x80x9cTopical anaesthesia of the skin by liposome-encapsulated tetracainexe2x80x9d Anesthesia and Analgesia 67:1079-1081; Patel (1985) xe2x80x9cLiposomes as a controlled-release systemxe2x80x9d,Biochemical Soc. Trans. 13:513-516; Wohlrab et al. (1987) xe2x80x9cPenetration kinetics of liposomal hydrocortisone in human skinxe2x80x9d Dermatologica 174:18-22].
Liposomes are microcapsules [diameters typically on the order of less than 0.1 to 20 xcexcm] that contain selected mixtures and can slowly release their contents in a sustained release fashion. Targeted liposomes or other capsule, particularly a time release coating, that dissolve upon exposure to oxygen, air, moisture, visible or ultraviolet [UV] light or a particular pH or temperature [see, e.g., U.S. Pat. No. 4,882,165; Kusumi et al. (1989) Chem. Lett. no.3 433-436; Koch Troels et al. (1990) Bioconjugate Chem. 4:296-304; U.S. Pat. No. 5,482,719; U.S. Pat. No. 5,411,730; U.S. Pat. No. 4,891,043; Straubinger et al. (1983) Cell 32:1069-1079; and Straubinger et a. (1985) FEBS Lttrs. 179:148-154; and Duzgunes et al. in Chapter 11 of the book CELL FUSION, edited by A. E. Sowers; Ellens et al. (1984) Biochemistry 23:1532-1538; Yatvin et al. (1987) Methods in Enzymology 149:77-87] may be used. Liposome formulations for use in baking [see, e.g., U.S. Pat. No. 4,999,208] are available. They release their contents when eaten or heated. Such liposomes may be suitable for intravenous or local administration.
Liposomes be prepared by methods known to those of skill in the art [see, e.g., Kimm et al. (1983) Bioch. Bioph. Acta 728:339-398; Assil et al. (1987) Arch Ophthalmol. 105:400; and U.S. Pat. No. 4,522,811, and other citations herein and known to those of skill in the art].
Liposomes that are sensitive to low pH [see, e.g., U.S. Pat. Nos. 5,352,448, 5,296,231; 5,283,122; 5,277,913, 4,789,633] are particularly suitable for use with alkaline agents. Upon contact with the low pH detergent or soap composition or a high pH composition, the contents of the liposome will be released. Other components, particularly Ca+ or the presence of dissolved O2 in the water will cause the components to glow as they are released. Temperature sensitive liposomes are also suitable for use in bath powders for release into the warm bath water.
c. Encapsulating Vehicles -Gelatin and Polymeric Vehicles
Macro or microcapsules made of gelatin or other such polymer that dissolve or release their contents on contact with air or light or changes in temperature may also be used to encapsulate components of the bioluminescence generating systems.
Such microcapsules or macrocapsules may also be conjugated to a targeting agent, e.g., an antibody, such that the bioluminescence generating components are delivered to the target by the antibody and then the components are released to produce a glow.
The aequorin system is particularly suitable for this application. It can be encapsulated in suspension or solution or as a paste, or other suitable form, of buffer with sufficient chelating agent, such as EDTA, to prevent discharge of the bioluminescence. Upon exposure of the capsule [microcapsule or macrocapsule] to moisture that contains Ca2+, such as in a buffer or blood, the released components will glow.
Thus, encapsulated bioluminescence generating components can be used in combination with a variety of targeting agents and thereby release the luciferase/luciferin, such as the Renilla system, which will light upon exposure to air], and other such items.
Other encapsulating containers or vehicles for use with the bioluminescence systems are those that dissolve sufficiently in water to release their contents, or that are readily opened when squeezed in the hand or from which the contents diffuse when mixed with a aqueous mixture. These containers can be made to exclude water, so that the bioluminescent system components may be desiccated and placed therein. Upon exposure to water, such as in an aqueous composition solution or in the atmosphere, the vehicle dissolves or otherwise releases the contents, and the components react and glow. Similarly, some portion less than all of the bioluminescence generating components may themselves be prepared in pellet form. For example, the component(s) may be mixed with gelatin or similar hardening agent, poured into a mold, if necessary and dried to a hard, water soluble pellet. The encapsulating containers or vehicles may be formed from gelatin or similar water soluble material that is biocompatible.
d. Endosomes and Vacuoles
Vehicles may be produced using endosomes or vacuoles from recombinant host cells in which the luciferase is expressed using method known to those of skill in the art [see, e.g., U.S. Pat. Nos. 5,284,646, 5,342,607, 5,352,432, 5,484,589, 5,192,679, 5,206,161, and 5,360,726]. For example, aequorin that is produced by expression in a host, such as E. coli, can be isolated within vesicles, such as endosomes or vacuoles, after protein synthesis. Using routine methods the cells are lysed and the vesicles are released with their contents intact. The vesicles will serve as delivery vehicles. When used they will be charged with a luciferin, such as a coelenterazine, and dissolved oxygen, such as by diffusion, under pressure, or other appropriate means.
e. Micronized Particles
The bioluminescence generating system components that are suitable for lyophilization, such as the aequorin photoprotein, the Renilla system, and the Vargula systems, can be micronized to form fine powder and stored under desiccating conditions, such as with a desiccant. Contact with dissolved oxygen or Ca2+ in the air or in a mist that can be supplied or in added solution will cause the particles to dissolve and glow.
3. Immobilized Systems
a. Matrix Materials
In some embodiments, it will be desirable to provide at least one component of the bioluminescence generating system linked to a matrix substrate, which can then be locally or systemically administered. The matrix substrate will be biocompatible. When desired, a mixture or mixtures(s) containing the remaining components, typically a liquid mixture is applied, as by pouring or spraying onto the matrix substrate, to produce a glow. For example, the aequorin photoprotein, including coelenterazine and oxygen, is linked to the substrate. When desired, a liquid containing Ca2+, such as tap water or, preferably, a liquid mixture containing the Ca2+ in an appropriate buffer, is contacted, such as by spraying, with the matrix with linked luciferase. Upon contact the material glows.
In other embodiments, the luciferase, such as a Vargula luciferase, is linked to the substrate material, and contacted with a liquid mixture containing the luciferin in an appropriate buffer. Contacting can be effected by spraying or pouring or other suitable manner. The matrix material is incorporated into, onto or is formed into an article of manufacture, such as surgical sponge or as part of a microbead.
The kits may also include containers containing compositions of the linked components which can be provided in a form, such as sprayed on as a liquid and air dried, that can be applied to the substrate so that the item can be made to glow again. Thus, kits containing a first composition containing the targeting agent and a luciferase or a luciferin or both and luciferin, and a second composition containing the remaining components. The item as provided in the kit can be charged with the first composition, such as having the composition applied and dried, or may require charging prior to the first use. Alternatively, the item may be sprayed with both compositions when desired to produce a glow.
It is understood that the precise components and optimal means for application or storage are a function of the selected bioluminescence system. The concentrations of the components, which can be determined empirically, are not critical, but must be sufficient to produce a visible glow when combined. Typical concentrations are as low as nanomoles/l, preferably on the order of mg/l or higher. The concentration on the substrate is produced when a composition containing such typical concentration is applied to the material. Again, such ideal concentrations can be readily determined empirically by applying the first composition, letting it dry, spraying the second composition, and observing the result.
The matrix material substrates contemplated herein are generally insoluble materials used to immobilize ligands and other molecules, and are those that used in many chemical syntheses and separations. Such matrices are fabricated preferably from biocompatible, more preferably from biodegradable materials. Such substrates, also called matrices, are used, for example, in affinity chromatography, in the immobilization of biologically active materials, and during chemical syntheses of biomolecules, including proteins, amino acids and other organic molecules and polymers. The preparation of and use of matrices is well known to those of skill in this art; there are many such materials and preparations thereof known. For example, naturally-occurring matrix materials, such as agarose and cellulose, may be isolated from their respective sources, and processed according to known protocols, and synthetic materials may be prepared in accord with known protocols. Other matrices for use herein may comprise proteins, for example carrier molecules, such as albumin.
The substrate matrices are typically insoluble materials that are solid, porous, deformable, or hard, and have any required structure and geometry, including, but not limited to: beads, pellets, disks, capillaries, hollow fibers, needles, solid fibers, random shapes, thin films and membranes. Thus, the item may be fabricated from the matrix material or combined with it, such by coating all or part of the surface or impregnating particles.
Typically, when the matrix is particulate, the particles are at least about 10-2000 xcexcM, but may be smaller or larger, depending upon the selected application. Selection of the matrices will be governed, at least in part, by their physical and chemical properties, such as solubility, functional groups, mechanical stability, surface area swelling propensity, hydrophobic or hydrophilic properties and intended use. For use herein, the matrices are preferably biocompatible, more preferably biodegradable matrices.
If necessary the support matrix material can be treated to contain an appropriate reactive moiety or in some cases the may be obtained commercially already containing the reactive moiety, and may thereby serve as the matrix support upon which molecules are linked. Materials containing reactive surface moieties such as amino silane linkages, hydroxyl linkages or carboxysilane linkages may be produced by well established surface chemistry techniques involving silanization reactions, or the like. Examples of these materials are those having surface silicon oxide moieties, covalently linked to gamma-aminopropylsilane, and other organic moieties; N-[3-(triethyoxysilyl)propyl]phthelamic acid; and bis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readily available materials containing amino group reactive functionalities, include, but are not limited to, para-aminophenyltriethyoxysilane. Also derivatized polystyrenes and other such polymers are well known and readily available to those of skill in this art [e.g., the Tentagel(copyright) Resins are available with a multitude of functional groups, and are sold by Rapp Polymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 and U.S. Pat. No. 5,292,814; see, also Butz et al. (1994) Peptide Res. 7:20-23; Kleine et al. (1994) Immunobiol. 190:53-66].
These matrix materials include any material that can act as a support matrix for attachment of the molecules of interest. Such materials are known to those of skill in this art, and include those that are used as a support matrix. These materials include, but are not limited to, inorganics, natural polymers, and synthetic polymers, including, but are not limited to: cellulose, cellulose derivatives, acrylic resins, glass, silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the like [see, Merrifield (1964) Biochemistry 3:1385-1390], polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides, rubber, silicon, plastics, nitrocellulose, celluloses, natural sponges. Of particular interest herein, are highly porous glasses [see, e.g., U.S. Pat. No. 4,244,721] and others prepared by mixing a borosilicate, alcohol and water.
Synthetic matrices include, but are not limited to: acrylamides, dextran-derivatives and dextran co-polymers, agarose-polyacrylamide blends, other polymers and co-polymers with various functional groups, methacrylate derivatives and co-polymers, polystyrene and polystyrene copolymers [see, e.g., Merrifield (1964) Biochemistry 3:1385-1390; Berg et al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) in Pept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198; Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al. (1979) Isr. J. Chem. 17:243-247; Kent et al. (1978) J. Org. Chem. 43:2845-2852; Mitchell et al. (1976) Tetrahedron Lett. 42:3795-3798; U.S. Pat. No. 4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat. No. 5,389,449]. Methods for preparation of such matrices are well-known to those of skill in this art.
Synthetic matrices include those made from polymers and co-polymers such as polyvinylalcohols, acrylates and acrylic acids such as polyethylene-co-acrylic acid, polyethylene-co-methacrylic acid, polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate, polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid, polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate, polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and those containing acid anhydride groups such as polyethylene-co-maleic anhydride, polypropylene-co-maleic anhydride and the like. Liposomes have also been used as solid supports for affinity purifications [Powell et al. (1989) Biotechnol. Bioeng. 33:173].
For example, U.S. Pat. No. 5,403,750, describes the preparation of polyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plant growth medium containing a hydrophilic polyurethane gel composition prepared from chain-extended polyols; random copolymerization is preferred with up to 50% propylene oxide units so that the prepolymer will be a liquid at room temperature. U.S. Pat. No. 3,939,123 describes lightly crosslinked polyurethane polymers of isocyanate terminated prepolymers containing poly(ethyleneoxy) glycols with up to 35% of a poly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producing these polymers, an organic polyamine is used as a crosslinking agent. Other matrices and preparation thereof are described in U.S. Pat. Nos. 4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992, 5,334,640, 5,328,603.
U.S. Pat. No. 4,162,355 describes a polymer suitable for use in affinity chromatography, which is a polymer of an aminimide and a vinyl compound having at least one pendant halo-methyl group. An amine ligand, which affords sites for binding in affinity chromatography is coupled to the polymer by reaction with a portion of the pendant halo-methyl groups and the remainder of the pendant halo-methyl groups are reacted with an amine containing a pendant hydrophilic group. A method of coating a substrate with this polymer is also described. An exemplary aminimide is 1,1-dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl compound is a chloromethyl styrene.
U.S. Pat. No. 4,171,412 describes specific matrices based on hydrophilic polymeric gels, preferably of a macroporous character, which carry covalently bonded D-amino acids or peptides that contain D-amino acid units. The basic support is prepared by copolymerization of hydroxyalkyl esters or hydroxyalkylamides of acrylic and methacrylic acid with crosslinking acrylate or methacrylate comonomers are modified by the reaction with diamines, aminoacids or dicarboxylic acids and the resulting carboxyterminal or aminoterminal groups are condensed with D-analogs of aminoacids or peptides. The peptide containing D-amino-acids also can be synthesized stepwise on the surface of the carrier.
U.S. Pat. No. 4,178,439 describes a cationic ion exchanger and a method for preparation thereof. U.S. Pat. No. 4,180,524 describes chemical syntheses on a silica support.
Immobilized Artificial Membranes [IAMs; see, e.g., U.S. Pat. Nos. 4,931,498 and 4,927,879] may also be used. IAMs mimic cell membrane environments and may be used to bind molecules that preferentially associate with cell membranes [see, e.g., Pidgeon et al. (1990) Enzyme Microb. Technol. 12:149].
These materials are also used for preparing articles of manufacture, surgical sponges soaps, and other items, and thus are amenable to linkage of molecules, either the luciferase, luciferin, mixtures of both. For example, matrix particles may be impregnated into items that will then be contacted with an activator.
Kits containing the item including the matrix material with or without the coating of the bioluminescence generating components, and compositions containing the remaining components are provided.
b. Immobilization and Activation
Numerous methods have been developed for the immobilization of proteins and other biomolecules onto insoluble or liquid supports [see, e.g., Mosbach (1976) Methods in Enzymology 44; Weetall (1975) Immobilized Enzymes, Antigens, Antibodies, and Peptides; and Kennedy et al. (1983) Solid Phase Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp. 253-391; see, generally, Affinity Techniques. Enzyme Purification: Part B. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad. Press, N.Y. (1974); Immobilized Biochemicals and Affinity Chromatography, Advances in Experimental Medicine and Biology, vol. 42, ed. R. Dunlap, Plenum Press, N.Y. (1974)].
Among the most commonly used methods are absorption and adsorption or covalent binding to the support, either directly or via a linker, such as the numerous disulfide linkages, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups, known to those of skill in art [see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog and Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such reagents; and Wong (1993) Chemistry of Protein Conjugation and Cross Linking, CRC Press; see, also DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Zuckermann et al. (1992) J. Am. Chem. Soc. 114:10646; Kurth et al. (1994) J. Am. Chem. Soc. 116:2661; Ellman et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:4708; Sucholeiki (1994) Tetrahedron Lttrs. 35:7307; and Su-Sun Wang (1976) J. Org. Chem. 41:3258; Padwa et al. (1971) J. Org. Chem. 41:3550 and Vedejs et al. (1984) J. Org. Chem. 49:575, which describe photosensitive linkers].
To effect immobilization, a solution of the protein or other biomolecule is contacted with a support material such as alumina, carbon, an ion-exchange resin, cellulose, glass or a ceramic. Fluorocarbon polymers have been used as supports to which biomolecules have been attached by adsorption [see, U.S. Pat. No. 3,843,443; Published International PCT Application WO/86 03840]. For purposes herein, the support material will be biocompatible (i.e., suitable for use in the body).
A large variety of methods are known for attaching biological molecules, including proteins and nucleic acids, molecules to solid supports [see. e.g., U.S. Pat. No. 5,451,683]. For example, U.S. Pat. No. 4,681,870 describes a method for introducing free amino or carboxyl groups onto a silica matrix. These groups may subsequently be covalently linked to other groups, such as a protein or other anti-ligand, in the presence of a carbodiimide. Alternatively, a silica matrix may be activated by treatment with a cyanogen halide under alkaline conditions. The anti-ligand is covalently attached to the surface upon addition to the activated surface. Another method involves modification of a polymer surface through the successive application of multiple layers of biotin, avidin and extenders [see, e.g., U.S. Pat. No. 4,282,2871; other methods involve photoactivation in which a polypeptide chain is attached to a solid substrate by incorporating a light-sensitive unnatural amino acid group into the polypeptide chain and exposing the product to low-energy ultraviolet light [see, e.g., U.S. Pat. No. 4,762,881]. Oligonucleotides have also been attached using a photochemically active reagents, such as a psoralen compound, and a coupling agent, which attaches the photoreagent to the substrate [see, e.g., U.S. Pat. No. 4,542,102 and U.S. Pat. No. 4,562,157]. Photoactivation of the photoreagent binds a nucleic acid molecule to the substrate to give a surface-bound probe.
Covalent binding of the protein or other biomolecule or organic molecule or biological particle to chemically activated solid matrix supports such as glass, synthetic polymers, and cross-linked polysaccharides is a more frequently used immobilization technique. The molecule or biological particle may be directly linked to the matrix support or linked via linker, such as a metal [see, e.g., U.S. Pat. No. 4,179,402; and Smith et al. (1992) Methods: A Companion to Methods in Enz. 4:73-78]. An example of this method is the cyanogen bromide activation of polysaccharide supports, such as agarose. The use of perfluorocarbon polymer-based supports for enzyme immobilization and affinity chromatography is described in U.S. Pat. No. 4,885,250]. In this method the biomolecule is first modified by reaction with a perfluoroalkylating agent such as perfluorooctylpropylisocyanate described in U.S. Pat. No. 4,954,444. Then, the modified protein is adsorbed onto the fluorocarbon support to effect immobilization.
The activation and use of matrices are well known and may be effected by any such known methods [see, e.g., Hermanson et al. (1992) Immobilized Affinity Ligand Techniques, Academic Press, Inc., San Diego]. For example, the coupling of the amino acids may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford.
Other suitable methods for linking molecules to solid supports are well known to those of skill in this art [see, e.g., U.S. Pat. No. 5,416,193]. These include linkers that are suitable for chemically linking molecules, such as proteins, to supports and include, but are not limited to, disulfide bonds, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups. These bonds can be produced using heterobifunctional reagents to produce reactive thiol groups on one or both of the moieties and then reacting the thiol groups on one moiety with reactive thiol groups or amine groups to which reactive maleimido groups or thiol groups can be attached on the other. Other linkers include, acid cleavable linkers, such as bismaleimideothoxy propane, acid labile-transferrin conjugates and adipic acid diihydrazide, that would be cleaved in more acidic intracellular compartments; cross linkers that are cleaved upon exposure to UV or visible light and linkers, such as the various domains, such as CH1, CH2, and CH3, from the constant region of human IgG, (see, Batra et al. (1993) Molecular Immunol. 30:379-386). Presently preferred linkages are direct linkages effected by adsorbing the molecule to the surface of the matrix. Other linkages are photocleavable linkages that can be activated by exposure to light [see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which linkers are herein incorporated by reference]. The photocleavable linker is selected such that the cleaving wavelength that does not damage linked moieties. Photocleavable linkers are linkers that are cleaved upon exposure to light [see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, which describes the use of a nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al. (1989) Makromol. Chem 190:69-82, which describes water soluble photocleavable copolymers, including hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer and methylrhodamine copolymer; Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which describes a cross-linker and reagent that undergoes photolytic degradation upon exposure to near UV light (350 nm); and Senter et al. (1985) Photochem. Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl chloride cross linking reagents that produce photocleavable linkages]. The selected linker will depend upon the particular application and, if needed, may be empirically selected.
These methods for linking molecules to supports may be adapted for use to link the targeting agents to the targeted agents.
F. Surgical Devices and Instruments
a. A Surgical Viewing Device for Visualizing Bioluminescent Neoplasia and Specialty Tissues
(1) Background
Monocular and binocular night vision devices have been developed to enable a viewer to observe objects at night and under low light conditions. These night vision devices may be battery powered, modular or solid-state, hand-held, such a telescope or night viewer, or mounted on a gun or helmet, such as a weapon sight or binoculars/goggles or other head gear (e.g., see U.S. Pat. Nos. 3,509,344; 4,449,783; 4,629,295; 4,642,452; 4,734,939; 4,822,994; 4,948,210; 4,953,963; 5,029,963; 5,084,780; 5,117,553; 5,146,077, 5,396,069; and, 5,535,053).
Several night vision devices are commercially available that detect low intensity visible and infrared light [e.g., monocular and binocular goggles, pocketscopes and viewers; Stano Components, Carson City, Nev.; Star-Tron Technology Corp., Pittsburgh, Pa., Excalibur Enterprises; RETRON night vision devices, Surveillance Technology Group, Mamaroneck, N.Y.; Princeton Instruments, MA.]. These night vision devices may be passive, relying only on ambient light, or active, where an external light source provides illumination for enhanced viewing.
In general, active night vision devices are contain of a power source and an infrared illuminator, e.g., a laser, that projects a beam of near infrared light [U.S. Pat. No. 4,948,210 describes an infrared zoom illuminator means]. Infrared and low intensity visible light is received from the surrounding environment through an objective camera lens that focuses and transmits images in the viewing area onto an image intensifier tube [e.g., GEN II and III image intensifiers are described in U.S. Pat. No. 5,146,077]. The GEN II and III intensifier tubes are commercially available (ITT Electro-Optical Products Division, Roanoke, Va.). The GEN III intensifier tube, preferred of the existing intensifier tubes, results in greater than a 50,000-fold amplification in light intensity (with xcx9c30-50% quantum efficiency between 600-700 nm) and a lifetime use of about 10,000 hours.
An image intensifier may contain a S-20 or S-25 photocathode, a photoemissive wafer that is extremely sensitive to low radiation levels of light in the 580-900 nm spectral range. The photocathode may be optionally coated with gallium arsenide, which increases the light amplification 4-fold (e.g., as available in the GEN III intensifier tube). The intensified image may be projected, for example, onto a phosphor screen of a photosensor, which converts the electronic emissions into visible light for viewing. The intensified images are viewed on the phosphor screen through an ocular lens assembly.
In addition, a microchannel plate (MCP) may be placed between the photocathode and the phosphor screen to enhance the brightness of the image. For example, 18 mm and 25 mm MCP second generation imager intensifiers are commercially available (Stano Components, Carson City, Nev.).
Alternatively, a charged coupled device (CCD) detector array may be used in association with the video camera/image intensifier system. In this instance, the intensified image may be transmitted through the CCD imager of a video camera which is connected to a display apparatus via a control unit [e.g., see U.S. Pat. No. 4,642,452].
(2) Surgical Vision Device
A surgical vision device useful for detecting low intensity visible light from bioluminescent neoplasia and specialty tissue is provided. The surgical vision device is analogous to the above-described night vision devices in that the image intensifier preferably receives or detects low intensity visible light, particularly wavelengths between 500-900 nm. Preferred for the in vivo detection of neoplasia and specialty tissue are wavelengths of visible light, preferably those wavelengths between 500-900 nm, more preferably green, red and near infrared wavelengths. In particularly preferred embodiments, the surgical night vision device is highly sensitive to low intensity visible red light.
Any night vision device that detects low intensity visible light is contemplated for use in the methods described herein. In addition, an existing vision device may be modified for use herein, such as by replacing the infrared-sensitive CCD imager or photocathode of the image intensifier, with a corresponding component that has an exceptionally high sensitivity to low intensity visible light. The GEN III image intensifier adequately detects light in the desired wavelengths, especially red and near infrared light (e.g., light wavelengths greater than xcex650 nm). The selection of the appropriate image intensifier or requisite modifications will depend on the wavelength and intensity of the light generated by the bioluminescent reaction which may be determined empirically by one of skill in the art using the teachings herein.
To minimize undesired wavelengths of light, an optical interference filter may also be employed in conjunction with these devices to allow only the desired wavelengths of light to pass through to the image intensifier or other equivalent imager. Such a filter may be included between the optical detector and the image intensifier to minimize the level of background light, i.e., wavelengths less than 500 nm or greater than 1000 nm.
The monocular and binocular night vision devices described in U.S. Pat. Nos. 5,396,069 or 5,535,053 are particularly suited for use in the methods described herein, particularly the modular apparatus described in U.S. Pat. No. 5,535,053. As described therein, a GEN III image intensifier (i.e., item 40 in FIG. 2) is contained in a tubular housing flanked on one end by an adjustable objective lens assembly and an adjustable ocular lens assembly attached to the opposite end of the housing. The intensified image produced by the GEN III intensifier tube is viewed on the phosphor screen through the adjustable ocular lens assembly. The GEN III image intensifier may be used or replaced with a modified image intensifier that has a higher sensitivity low intensity visible light.
(3) Methods of Use
The surgical vision device provides the surgeon a means for visualizing the image of a tumor or specialty tissue from the low intensity visible light produced at the target site from a selected bioluminescence generating system. For example, a targeting agent conjugated to a component of the bioluminescence generating system, preferably a luciferase, may be administered to a patient followed by the remaining components of the bioluminescence generating system. After exposure of the target area for surgical viewing, the image of the glowing tumor or specialty tissue is captured by the objective lens and focused onto the photocathode tube of a GEN III or equivalent image intensifier. In a dark, sterile room, the glowing image is readily detected against the dark background of the body cavity and the image projected on the phosphor screen is viewed through the ocular lens assembly. Metastatic tumors may also be rapidly identified by visual inspection of the exposed surrounding tissues and organs. Surgical removal of the target may occur in the absence or presence of the assistance of the surgical vision device.
In particularly preferred embodiments, the surgical vision device is used for the detection of deep tumors and metastases using a bioluminescence generating system that produces red light at the target site. Presently preferred are the bioluminescence generating systems isolated from Aristostomias, such as A. scintillans, Pachystomias and Malacosteus, such as M. niger, all of which produce red light [i.e., light of a wavelength between 650 and 750 nm, peaking at 705 nm; Widder et al. (19??) Science News 150(14): 214].
In alternative embodiments, blue or green light may produced at the target site using a bioluminescence generating system, e.g., using Renilla bioluminescence generating system, and converted to red light by further including a fluorescent protein, such as a phycobiliprotein, which converts green light to red light. The concentration of the bioluminescence generating components necessary to generate red light of sufficient intensity to be detected by the surgical vision device will vary but may be determined empirically by one of skill in the art using methods described herein and those known to those of skill in the art.
b. Miniature Surgical Imaging Instruments
(1) Background
Optical images of the interior of a living body may also be generated by a surgeon using a surgical imaging instrument. Examples of instruments used for surgically viewing the internal organs of a patient include, but are not limited to, laparoscopes, endoscopes, cytoscopes, arthoscopes and thorascopes [e.g., see U.S. Pat. Nos. 5,511,564; 5,311,859; 5,313,306 and 5,331,950].
The application of these instruments requires only a minimally invasive surgical procedure. In general, the surgeon makes a small incision in the patient nearby the targeted viewing area into which the elongated tube of the instrument is inserted. The instruments are guided through the body to the viewing area by the surgeon or by remote controlled means [e.g., see U.S. Pat. No. 5,540,649 describing a device for remotely controlling surgical instruments]. Light is transmitted from a light source through the insertion tube using a fiber optic bundle to illuminate internal objects of the body cavity. The illuminated images are focused by a lens housed at the distal end of the insertion tube onto an image forming surface of a CCD imager or other equivalent imager. The optical image is then converted to an electric output signal that is displayed on a video monitor, LED monitor or phosphor screen.
In addition, a wide angled lens may be used to increase the available viewing area, and any image distortion from such lenses may be optically corrected using an omniview transformation system, which allows for multiple simultaneous images to be outputted from a single location [e.g., see U.S. Pat. No. 5,313,306].
(2) Surgical Viewing Instruments for Detecting Bioluminescent Neoplasia and Specialty Tissue
Surgical instruments for detecting bioluminescent neoplasia and specialty tissue are provided herein. More particularly, surgical instruments containing miniaturized imagers that are specifically designed to detect bioluminescence inside the body cavity of a patient are provided herein.
Any surgical imaging device capable of detecting emissions generated from a bioluminescent generating system is contemplated for use in the methods and EXAMPLES provided herein. For example, surgical instruments that detect emissions generated throughout the human scotopic and photopic range, e.g., ultraviolet through infrared wavelengths, are contemplated for use herein. In preferred embodiments the instruments are highly sensitive to broad spectrum visible light, and in more preferred embodiments, the instruments are highly sensitive to narrow ranges visible light, particularly blue, green or red wavelengths.
In especially preferred embodiments, surgical viewing instruments contain a modified CCD imager or other equivalent imager that is useful for the detection of bioluminescence. Particularly preferred instruments are those described in U.S. Pat. No. 5,313,306 (endoscope), U.S. Pat. Nos. 5,311,859 and 5,331,950 (laparoscopes). For example, U.S. Pat. No. 5,331,950 describes a video laparoscope assembly in which a miniature camera, containing a lens assembly, focuses an image of a target onto an image forming surface of CCD video camera imager. The intensified image is converted to an output signal which is sent to a video processing unit. The reproduced image is displayed on an accompanying video monitor.
Existing CCD imagers and other equivalent image intensifiers may be used in the methods that are sensitive to low intensity visible and infrared light. These CCD imagers are commercially available and well known to those of skill in the art (e.g., Tektronics TEK Nos. 25, 27 and 32 CCD imager intensifiers; Generation II and III image intensifier tubes, ITT Electro Optics Division, Roanoke, Va.; cooled CCD arrays and image intensifiers, Princeton Instruments, Inc., Fremont, Calif.; SenSys 1400 and 1600 digital cameras, Photometrics, Ltd; Model 221, Javelin Electronics and, 18 and 25 mm MCP second generation image intensifier tubes, Stano Components, Inc, Carson City, Nev.). Any of these imager intensifiers may be used in the apparatus described herein.
The selection of the appropriate CCD imager for use in the methods herein will depend on means of visualizing the tumor and the wavelength and intensity of the light generated by the bioluminescent reaction. These parameters may be determined empirically by one of skill in the art using the teachings provided herein. For the detection of extremely low levels of light emission, the surgical instrument may optionally contain an image intensifier tube, e.g., a GEN III image intensifier tube or red-sensitive Model 221 image intensifier, and the intensified image visualized on a phosphor screen or other equivalent display means.
Using these surgical instruments, an external light source is not required for viewing the bioluminescent target because the tumor or specialty tissue targeted for surgical removal glows in the absence of excitation by an external illumination source. When necessary, a light source may be used that generates broad spectrum light or monochromatic light of narrow spectral bands. In addition, the color of the broad spectrum light may be filtered to reduce background and light scattering. Color wheel filters adaptable for use in these instruments are known [e.g., see U.S. Pat. No. 4,523,224]. Alternatively, in order to minimize undesired wavelengths of light from reaching the CCD imager or intensifier tube, an appropriate optical interference filter may be placed between the video camera and the imager.
In a preferred embodiment, the CCD imager of the surgical instrument is highly sensitive to visible light, preferably blue or green light [xcex400-600 nm]. A tumor-specific targeting agent, e.g., an antibody directed against a colon cancer specific-antigen, is coupled to a component of the Aequorin or Renilla bioluminescence generating system, preferably the photoprotein or luciferase, respectively. In the presence of the remaining components, bluish-green light is produced at the site of the tumor. The remaining components, i.e. an appropriate substrate and/or any necessary activators, may be added intravenously or using a laparoscope or trocar that allows for local administration of fluid to the target site through the insertion tube [e.g., see International Patent Application No. WO 95/32012]. A laparoscope that contains a CCD imager that is highly sensitive to low intensity visible light in these wavelengths, is used by the surgeon to detect and visualize the image of the glowing tumor. The target tissue may be removed surgically or removed using a medical ablation device [e.g., see U.S. Pat. Nos. 5,486,161 and 5,511,564 describing laparoscopic medical ablation devices].
In another preferred embodiment, the CCD imager of the surgical imaging instrument is replaced with an imager containing a CCD array that is highly sensitive to low intensity red light [e.g., xcex650-750 nm]. Wavelengths of red and near infrared light of about 700-1300 nm are known to easily penetrate through the skull, soft tissue and into bone [e.g., see U.S. Pat. No. 4,281,645]. In this embodiment, a tumor-specific targeting agent is coupled to a component of a red emitting bioluminescent generating system, e.g., Aristostomias, and the remaining components of the reaction are added as described above. Red light emitted from the tumor is detected using the modified laparoscope or endoscope. The glowing image of the tumor may be visualized through surrounding and nearby tissues, even in deep tissue tumors such as breast cancer, eliminating the need to surgically view the targeted tissue.
c. Computer Tomographs
(1) Background
A computer tomograph is a non-invasive, medical imaging device capable of generating prescribed sectional pictures of the interior of a living body. These sectional images, i.e., computer tomograms, are arranged in different planes from which a three dimensional computer image of the internal organs and tissues of the body may be constructed.
In general, a computer assisted tomograph, e.g., an X-ray CT scanner, is composed of a patient support, an X-ray radiator and detector array capable of being rotated 360 degrees around the patient axis and a computer processing unit for image re-construction. In preparing computer tomograms, a subject is placed horizontally on the patient support and irradiated with a narrow beam of X-rays. The X-ray radiator and detector array, fixed in space relative to one another, are rotated 360 degrees around the patient. The circumferential output signals from the detector array are relayed to a computer processing unit which reconstructs an image of the target. The reproduced image is viewed on a video monitor or other suitable display means [e.g., see U.S. Pat. Nos. 5,113,077; 5,315,628; 5,533,082].
In addition to images generated using X-rays, apparatus for optically inspecting the human body using light are also known. For example, spectrophotometric methods and apparatus for optically monitoring biological processes, such as blood metabolism, are known [e.g., see U.S. Pat. Nos. 4,281,645; 5,148,022; 5,337,745; 5,413,098; International Patent Application Publication No. WO 93/13395; Amato (1992) Science 258:892-893]. Furthermore, optical tomography using laser-emitting apparatus that pulse wavelengths of near infrared light has been employed to visualize images of phantom objects placed in tissue and also the internal organs of animals [Benaron et al. (1994) in Oxygen Transport to Tissue, Ed. Hogan et al., pp. 207-222 Plenum Press, NY].
Conventional tomographic and spectrophotometric apparatus require an external source of radiation for image detection and construction. For example, traditional computer tomography requires an irradiation source for generating X-rays. Optical tomographs require one or more lasers to generate a narrow beam of light in the near infrared region of the spectrum to minimize scattering and absorption of photons by tissues. Computer software containing algorithms for forward and inverse problems of image re-construction in optical tomography has been developed, such as time-of-flight absorbance [TOFA; e.g., see Benaron et al. (1993) Science 259:1463-1466; Benaron et al. (1994) in Oxygen Transport to Tissue, Ed. Hogan et al., pp. 207-222 Plenum Press, NY] or time-resolved optical absorption and scattering tomography [TOAST; e.g., Arridge et al. (1991) Proc. SPIE 1431:204-215; Arridge et al. (1993) Proc. SPIE 2035:218-229].
(2) Computer Apparatus for the Non-invasive Detection of Bioluminescent Neoplasia and Specialty Tissue
A computerized apparatus and non-invasive methods for optically inspecting the interior of a living body are provided herein. More particularly, a computerized optical imaging apparatus and non-invasive methods for optically imaging bioluminescent neoplasia and specialty tissues are provided herein.
It is to be appreciated that any apparatus that is capable of detecting electromagnetic radiation generated from a bioluminescent reaction and calculating an image of the bioluminescent target is contemplated for use in the methods described herein. For example, the computerized optical imaging apparatus may be analogous to a computer tomograph except that bioluminescence is used as the radiation source and an optical detector as the image detector. Image reconstruction may be performed using a computer processing unit containing software that uses algorithms and principles employed in X-ray CT scanners and optical computer tomography, such as time-of flight optical imaging, convolution integration and Fourier analysis [e.g., see Benaron et al. (1993) Science 259: 1463-1466; U.S. Pat. Nos. 5,113,077; 5,315,628; 5,533,082].
Alternatively, the apparatus may contain any optical detector known to those of skill in the art capable of detecting electromagnetic radiation through tissue or other biological medium. Visible light may be detected directly through animal tissue using a photon detector or digital camera.[e.g., see Travis (1996) Science News 150:220-221]. Thus, the instant apparatus may contain an array of digital cameras may be used to detect light emission from the bioluminescent neoplasia or specialty tissue. In this exemplary embodiment, digital cameras may be placed at any angle or any position nearby a targeted viewing area or, alternatively, a complete scan of the entire patient may be performed. The electromagnetic radiation detected by the digital camera may be relayed to a computer processing unit and computer image of the bioluminescent neoplasia or specialty tissue is re-constructed based from the intensity of the detected light emissions using one or more of the above-described computer algorithms.
By moving one or more digital camera, several computer images may be produced and overlaid upon one another to generate a three dimensional image of the bioluminescent neoplasia or specialty tissue. The imaging of the bioluminescent tumors or specialty tissues may be also be performed by overlaying against images obtained using other non-invasive diagnostic imaging methods. Particularly preferred is the projection of bioluminescent neoplasms on images obtained using standard ultrasound techniques, such as sonograms [e.g., see U.S. Pat. Nos. 5,575,288; 5,619,995; 5,619,999; 5,622,172; 5,630,426].
In preferred embodiments, the optical imaging apparatus is capable of detecting low intensity visible light emissions from the interior of a patient. One or more digital camera is used to detect light output from the bioluminescent target, and the detected images are relayed to a computer processing unit that generates an image of the bioluminescent neoplasia or specialty tissue. In especially preferred embodiments, the optical detector of the apparatus is highly sensitive to wavelengths of low intensity red and near infrared light, which wavelengths are known to undergo the least amount of scattering through tissue or other biological medium.
G. Photodynamic Therapy
1. Background
The bioluminescent targeting agent conjugates may also be used the detection and treatment of neoplasia in conjunction with photodynamic therapy techniques. These techniques involve the administration of a photosensitizing drug to a patient. The drugs or chemicals subsequently localize in neoplastic cells which are then illuminated from an external source with light of an appropriate wavelength to activate the drug or chemicals. This photoactivation results in photochemical reactions in the neoplastic tissue that ultimately cause cytotoxic injury and/or death of the targeted tissue.
A wide variety of compounds suitable for use as photosensitizing drugs are commercially available or may be prepared using methods known to those of skill in the art. For example, the compounds may be derived from natural sources (e.g., porphyrins, chlorins and purpurins) or from known chemicals originating in the dyestuffs industries (e.g., cyanine dyes). Specific examples of photosensitizing drugs include, but are not limited to, phyhalocyanines (merocyanine 540), substituted purpurines, xanthenes (Rhodamine 1236GandB), cationic cyanine dyes, chlorine polymers, chalcogenapyrylium dyes containing selenium or tellurium atoms in the chromophore, phenothiazolium derivatives, benzophenoxoniums (Nile Blue A) and triarylmethanes (Victoria Blue BO) [e.g., see U.S. Pat. Nos. 4,861,876; 4,961,920; 5,132,101; 5,179,120; 5,189,029; 5,344,928; 5,409,900; 5,433,896; 5,446,157; 5,532,171 and International Patent Application Publication Nos. WO 95/24930 and WO 96/31451].
The photosensitizing drugs may be formulated, for example, for topical, local, parenteral or systemic intravenous administration. In addition, the specificity of the photosensitizing drug for neoplastic cells can also be increased by conjugation to a known anti-tumor targeting agent. For example, the photosensitizing drug may be conjugated to an antibody or other peptide. The method of conjugating a porphyrin dye to an anti-cancer antigen antibody is known [e.g., see European Patent Application No. EP 252683].
The exact mechanism used by the above-mentioned chemicals to destroy neoplastic cells upon exposure to an external excitatory light source is currently unknown. There are two generally proposed mechanisms by which photosensitizing drugs are chemically altered upon illumination. The first mechanism typically involves hydrogen atom abstraction from the drugs, thereby producing free radicals that react with organic products or oxygen, which results in biochemical destruction of the neoplastic cells. The other reaction mechanism normally involves energy transfer from the electronically excited drugs to oxygen producing a singlet oxygen. The singlet oxygen reacts with a variety of substrates to produce oxygenated products in combination with superoxide ions that results in disruption of the target cell wall or mitochondria and destruction of the neoplastic cell.
External light sources, such as white light sources and lasers, are typically used as the radiation source. The time and duration and repetition of irradiation varies depending on the particular photosensitizing compound selected, the means of administration and the neoplasia to be treated. For example, irradiation takes place not less than one hour nor more than four days after parenteral administration of a porphyrin derivative; whereas radiation may commence as soon as 10 minutes after topical application of the same or similar compound [e.g., see U.S. Pat. No. 5,409,900]. Photodynamic therapy is usually initiated about 3 to 48 hours after administration of the photosensitizing drug and may be performed using invasive as well as non-invasive surgical procedures. Apparatus for irradiating living cells are well known to those of skill in the art [e.g., see International Patent Application Publication No. WO 96/24406].
Not all neoplasia are suitable candidates for photodynamic therapy. Nevertheless, photodynamic therapy has been used to treat bladder, bronchial, bone marrow and skin tumors as well as severe psoriasis [e.g., see U.S. Pat. No. 5,179,120]. Specific examples of skin malignancies include basal and squamous cell cancers, malignant melanoma, Kaposi""s sarcoma, mycosis, fungoides, metastatic epidermoid, and recurrent breast cancer. Head and neck cancers, such as nasopharyngeal, tongue and other oropharyngeal tumors may also be treated. Other examples of neoplastic tissue that may be treated are endobronchial cancer (such as adenocarcinoma or small cell carcinoma), esophageal cancers, gynecologic tumors (such as cervical carcinoma, vaginal cancer and vulvar malignancies) as well as brain tumors (such as glioblastoma and astrocytoma) and metastatic malignancies [e.g., see U.S. Pat. No. 5,446,157].
2. Methods of Using Photodynamic Therapy in Conjunction with Bioluminescent Targeting Agents
The photosensitizing drugs used in photodynamic therapy may also be used in methods with the bioluminescent targeting agent conjugates, as described herein, in the detection and treatment of neoplasia and specialty tissue. These methods differ from traditional photodynamic therapy in that light required to activate the photosensitizing drug is generated in situ by a bioluminescent reaction. Thus, irradiation of the drug occurs directly at the surface of the neoplastic cell by light emitted by the targeting agent-luciferase conjugate, which has been localized on the surface of the targeted cell through the specific interaction of the targeting agent and the target. Therefore, the present method may be used with a wide variety of compounds and photodynamic therapies, and provides non-invasive surgical procedures for treating a diverse array of neoplasia.
For practice in the methods, an effective amount of a photosensitizing drug may be administered to a patient successively or simultaneously with an anti-tumor antigen antibody conjugated to one or more component of a bioluminescence generating system, preferably a luciferase. After allowing an amount of time sufficient for binding of the antibody conjugate to the neoplastic tissue and subsequent sequestration of the photosensitizing drug by the neoplastic tissue, e.g., 10 min to 24 hr, the remaining components of the bioluminescent reaction, e.g., luciferin and any necessary activators, are administered to initiate the bioluminescent reaction. Light production activates the photosensitizing drug resulting the death of the targeted neoplastic cell.
The bioluminescence generating system used to activate the photosensitizing drug will vary depending on the biochemical properties of the drug [e.g., the absorption maxima] and the type of neoplasia to be detected or treated. The selection of the drug and bioluminescence system may be determined empirically based on the teachings herein. In addition, the targeting agent may also include the use of a fluorophore or fluorescent protein, e.g., GFP, to alter the wavelength of the emitted light to provide a wider range of wavelengths for treatment.
This method is particularly advantageous for detecting and treating metastatic tumors using photodynamic methodologies. For example, metastases may be located quite distant from the primary site of irradiation used in traditional photodynamic techniques and, thus, metastatic regions may not receive a sufficient dose of irradiation for photoactivation of the drug. In the instant method, light may be continuously produced at any location in which the targeting agent binds. Therefore, sufficient quantities of light may be generated throughout the body to eliminate those neoplastic cells that have metastasized from the location of the primary tumor.
Presently preferred compounds for use in the methods herein are those that have absorption maxima between 400 and 900 nm wavelengths and particularly preferred bioluminescence generating systems are those that emit wavelengths of light greater than 500 nm, Aristostomias and Malecostus. In addition, methods using photodynamic therapy with one or more fluorescent protein and the bioluminescent generating systems of Renilla and the photoprotein aequorin are also preferred.
H. Practice of the Methods
Among the embodiments herein, are those in which a bioluminescence generating component, preferably a luciferase, is conjugated to an antibody directed against a human tumor antigen. A patient is administered a pre-operative dose of the antibody-luciferase conjugate, which binds to the tumor antigen. During the surgical procedure, the final components, i.e., a luciferin and any activators, are applied topically to the suspected area of the neoplasia, causing the tumor-antigen expressing cells to be illuminated. The illuminated area may be detected visually or using one of the above-described surgical instruments.
In the case of melanoma, for example, the antibody-luciferase conjugate may be applied topically in a lotion, ointment, cream or other such formulation, and the unbound and non-specifically bound antibody conjugate removed by washing or administered i.c.v or i.p. The remaining bioluminescence-generating components are topically applied to visualize the location and margins of the neoplastic melanoma cells for surgical removal.
The light intensity of the bioluminescent reaction may be amplified by coupling more than a single luciferase molecule to a microcarrier (e.g., microparticles or microbeads). The luciferase-coupled microcarrier may be covalently or non-covalently linked to a target agent and injected into an animal or human patient. Upon binding to the target, the remaining components of the bioluminescent reaction are added and the light production from the tumor or specialty tissue will be greatly enhanced compared to a targeting agent-luciferase protein conjugate (e.g., an aequorin-Mab conjugate, see U.S. Pat. No. 5,486,455).
Using the coupling methods described herein, a set of microcarriers may be designed containing one or more type of luciferase for the rapid production of a single targeting agent linked to one or more luciferase each having different biochemical properties (i.e., emit light of a different wavelength). In addition, more than one of the bioluminescence generating components may be coupled to the microcarrier. For example, aequorin or Renilla luciferase may be coupled, concurrently or successively, with a GFP, which absorbs light of one wavelength (xcexmax=480 nm) and emits light of a different wavelength (xcexmax=509 nm). Thus, Renilla luciferase bound to a microcarrier would emit blue light whereas a microcarrier containing Renilla luciferase and GFP would emit green light. Alternatively, the coupling a component of the Aristostomias, Pachystomias or Malacosteus bioluminescence generating system to a microcarrier would result in the production of red light.
The targeting agent may be conjugated to one or more components of the bioluminescence generating system contained on the microcarrier through a biotin-streptavidin complex. The interaction between avidin/streptavidin and biotin is well documented and has been exploited as a research tool across several scientific disciplines (e.g., see Bayer and Wichek (1980) The Use of Avidin/Biotin Complex as a Tool in Molecular Biology. Meth. Biochem. Anal. 26, 1-45).
The coupling of proteins and small molecules to biotin is well known and bioluminescence generating components are amendable to biotinylation. For example, biotinylated aequorin has been reported (Stultz et al. (1992) Use of Recombinant Biotinylated Apoaequorin from Escherichia coli. Biochemistry 31, 1433-1442; Smith et al. (1991) A Microplate Assay for Analysis of Solution Phase Glycosyltransferase Reactions: Determination of Kinetics Constants. Anal. Biochem. 199, 286-292). In addition, reagents are commercially available for coupling biotin to primary amines, sulfhydryl groups, carboxyl groups and the side chains of the amino acids tyrosine and histidine (see Pierce Catalog, pp. T123-T154, 1994). Kits designed for biotinylating compounds are also commercially available (e.g., Boehringer Mannheim Corp., Indianapolis, Ind.).
In addition, streptavidin matrices useful as microcarriers may be purchased or synthesized starting with the matrices described in Section 3(C)(3)(a) herein. Thus, the streptavidin-biotin coupling method is amendable to any targeting agent that may be coupled to biotin and any streptavidin matrix suitable as a microcarrier that may be coupled to at least one or more luciferase molecule.
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.